Humanized antibodies to ganglioside GM2

ABSTRACT

Chimeric human antibody expression vectors are constructed by inserting the antibody heavy chain variable region-encoding cDNA and antibody light chain variable region-encoding cDNA isolated from hybridomas producing a mouse or rat monoclonal antibody reacting with the ganglioside GM 2  respectively into an expression vector for use in animal cells which contains the human antibody heavy chain constant region- or human antibody light chain constant region-encoding cDNA. The expression vectors are introduced into animal cells and the transformant thus obtained is cultured for the production of a chimeric human antibody reacting with the ganglioside GM 2 . In contrast to mouse monoclonal antibodies, the chimeric human antibodies of the invention will not cause anti-mouse immunoglobulin antibody production in the patient&#39;s body but shows a prolonged blood half-life, with a reduced frequency of adverse effects, so that it can be expected to be superior to mouse monoclonal antibodies in the efficacy in the treatment of human cancer, for instance.

FIELD OF THE INVENTION

The present invention relates to humanized antibodies reacting with theganglioside GM₂. The humanized antibodies do not cause production ofanti-mouse immunoglobulins in the patient's body as compared with mousemonoclonal antibodies, hence the incidence of adverse effects possiblycaused by them is much lower, their blood half-lives are longer and,further, their anti-tumor effector effect is greater. Therefore, thehumanized antibodies are expected to produce improved therapeuticeffects as compared with mouse monoclonal antibodies.

BACKGROUND OF THE INVENTION

When administered to humans, mouse antibodies are generally recognizedas foreign matters, inducing production of anti-mouse immunoglobulinantibodies in the human body. It is known that the former antibodiesreact with the latter antibodies to produce adverse effects J. Clin.Oncol., 2, 881 (1984); Blood, 65, 1349 (1985); J. Natl. Cancer Inst.,80, 932 (1988); Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)! and thatthe mouse antibodies undergo rapid clearance J. Nucl. Med., 26, 1011(1985); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 937 (1988)!,thus showing only a reduced efficacy J. Immunol., 135, 1530 (1985);Cancer Res., 46, 6489 (1986)!. Attempts have been made to solve theseproblems by deriving, from mouse monoclonal antibodies, chimeric humanantibodies or CDR (complementarity determining region)-transplantedantibodies (reshaped antibodies) using gene engineering technique. In ahuman chimeric antibody, the variable regions thereof are of mouseorigin and the constant regions thereof are of human origin Proc. Natl.Acad. Sci. U.S.A., 81, 6851 (1984)! and it is reported that whenadministered to humans, said antibody causes litte human anti-mouseimmunoglobulin antibody production, its blood half-life being 6-foldlonger Proc. Natl. Acad. Sci. U.S.A., 86, 4220 (1989)!. TheCDR-transplanted antibodies are antibodies resulting from replacement ofthe CDRs in a human antibody alone with the CDRs from an animal otherthan the human Nature, 321, 522 (1986)! and, in an experiment withmonkeys, such antibodies showed reduced immunogenicity and 4- to 5-foldhigher serum half-lives as compared with mouse antibodies J. Immunol.,147, 1352 (1991)!.

As regards the cytocidal activity of antibodies, it is reported that theFc region of a human antibody is more potent in activating humancomplement and human effector cells than the Fc region of a mouseantibody. Thus, for instance, a chimeric antibody derived from a mousemonoclonal antibody to the ganglioside GD₂ and containing a humanantibody Fc region enhances the human effector cell-mediated antitumoreffect J. Immunol., 144, 1382 (1990)!. Similar results are reported forCDR-transplanted antibodies Nature, 332, 323 (1988)!. Such resultsindicate that, for clinical use, humanized monoclonal antibodies arepreferred to mouse monoclonal antibodies.

The antibody classes include IgA, IgM, IgG, IgD and IgE and, in mice,the class IgG includes four subclasses, namely IgG₁, IgG₂ a, IgG₂ b andIgG₃ (in humans, IgG₁, IgG₂, IgG₃ and IgG₄). When antigens areadministered to animals, the antibodies produced mostly belong to theclasses IgM or IgG. IgG molecules have a molecular weight of about160,000 daltons and a dimeric structure and are relatively easy tohandle. IgM molecules are large with a molecular weight of about 900,000daltons and occur in the form of a complicated pentameric structurecoupled with the joining (J) chain, hence they have the followingdrawbacks: they are difficult to purify; they tend to agglutinate, henceare difficult to store; they are readily inactivated by partialdecomposition in the presence of a protease, hence it is difficult toprepare Fab fragments; and they lose their binding activity in manyinstances upon chemical modification, for example chemical binding of ananticancer agent or a toxin J. W. Goding: Monoclonal Antibodies:Principles and Practice, Academic Press, 1986!. As to which are superiorin therapeutic effect against cancer, IgG class monoclonal antibodies orIgM class monoclonal antibodies, reference may be made to a detailedstudy made by Bernstein et al. using an IgG class monoclonal antibodyand an IgM class monoclonal antibody to the lymphocyte Thy-1 antigenMonoclonal Antibodies, edited by R. H. Kennet, T. J. Mckearn and K. B.Bechtol, Plenum Press, 1980, p. 275!. According to the reference, an IgGclass monoclonal antibody and an IgM class monoclonal antibodycomparable in terms of reactivity to Thy-1 antigen-positive lymphocytes,were compared in terms of antitumor effect. While the IgM monoclonalantibody was superior in in vitro complement-dependent antitumor effect,the IgG class monoclonal antibody showed a significant antitumor effectin in vivo antitumor effect in cancer-bearing mice, with no antitumoreffect being observed with the IgM class monoclonal antibody. It wasfurther revealed that, as compared with the IgG class monoclonalantibody, the IgM class monoclonal antibody showed a very shorthalf-life in the blood after administration, in an isotope-labeled form,to mice. These results indicate that the monoclonal antibodies to beused clinically in humans should preferably be of the IgG class.

Gangliosides, a class of glycolipids, are constituents of animal cellmembranes. These molecules are composed of a carbohydrate chain, whichconstitutes a hydrophilic side chain, and sphingosine and a fatty acid,which constitute hydrophobic side chains. It is known that theganglioside species expressed and the amount thereof differ between cellspecies, organ species, and animal species, among others. Furthermore,it has been reported that the ganglioside expressed changedquantitatively and qualitatively during the process of cancerdevelopment Cancer Res., 45, 2405 (1985)!. For example, expression ofthe gangliosides GD₂, GD₃ and GM₂ has been reported in neuroblastoma,lung small cell carcinoma, and melanoma, which are highly malignantneural ectodermal tumors J. Exp. Med., 155, 1133 (1982); J. Biol. Chem.,257, 12752 (1982); Cancer Res., 47, 225 (1987); ibid., 47, 1098 (1987);ibid., 45, 2642 (1985); Proc. Natl. Acad. Sci. U.S.A., 80, 5392 (1983)!.

GM₂, one of the gangliosides that are sialic acid residue containingglycolipids, occurs only in trace amounts in normal cells but is foundin increased amounts in cancer cells in lung small cell carcinoma,melanoma, neuroblastoma, etc. Monoclonal antibodies to GM₂ areconsidered to be useful in the treatment of such cancers Lancet, 48,6154 (1988)!. However, those monoclonal antibodies to GM₂ that have sofar been reported are of the human IgM class or of the rat IgM, mouseIgM or mouse IgG class Cancer Res., 46, 4116 (1986); Proc. Natl. Acad.Sci. U.S.A., 79, 7629 (1982); Cancer Res., 48, 6154 (1988); J. Biol.Chem., 264, 12122 (1989)!.

Anti-ganglioside GM₂ monoclonal antibodies, if produced in the form ofhumanized antibodies, for example chimeric human antibodies orCDR-transplanted antibodies, which are not expected to induce anti-mouseimmunoglobulin antibody production in the patient's body, producereduced adverse effects and show a prolonged blood half-life and anenhanced antitumor effector effect. These antibodies are thus expectedto be superior in therapeutic effect to the corresponding mousemonoclonal antibodies.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide humanized antibodiesto the ganglioside GM₂ (hereinafter, "humanized anti-GM₂ antibodies")which are useful in the treatment of cancers of neural ectodermalorigin, among others.

The present inventors prepared the antibody heavy chain (hereinafter, "Hchain") variable region (hereinafter "V_(H) ") cDNA and light chain(hereinafter, "L chain") variable region (hereinafter, "V_(L) ") cDNAsfrom mRNAs isolated from the hybridomas KM750 and KM796, described inEP-A-0 508 472.

These hybridomas produce IgG₃ class mouse monoclonal antibodies to theganglioside GM2. V_(H) and V_(L) cDNAs were also prepared from mRNAsisolated from the hybridoma KM603, which produces an IgM class ratmonoclonal antibody to the ganglioside GM₂. Chimeric human antibodyexpression vectors were constructed by inserting the cDNA into anexpression vector containing human antibody H chain constant region(hereinafter, "C_(H) ") or human antibody L chain constant region(hereinafter, "C_(L) ") encoding sequences. Such vectors were thenintroduced into animal cells to effect the production ofanti-ganglioside GM₂ chimeric human antibodies. Among the chimericantibodies produced, the anti-ganglioside GM₂ chimeric human antibody,KM966, was found to react with the ganglioside GM₂ and show cytocidalactivity. The H chain variable region of KM966 contains an amino acidsequence segment as defined by SEQ ID NO:1 and includes the 1st to 120thamino acids of that sequence and the L chain variable region of KM966contains an amino acid sequence segment as defined by SEQ ID NO:2 andincludes the 1st to 107th amino acids of said sequence. The presentinvention is based, at least in part, on these findings.

The present invention thus relates to a humanized antibody reacting withthe ganglioside GM₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates plasmids, pKM796H1 and pKM796L1.

FIG. 2 illustrates plasmids, pKM750H1 and pKM750L1.

FIG. 3 illustrates plasmids, pKM603H1 and pKM603L1.

FIG. 4 shows a construction scheme for a plasmid, pAGE147.

FIG. 5 shows a construction scheme for a plasmid, pAGE148.

FIG. 6 shows a construction scheme for plasmids, pChi796HM1 andpChi750HM1.

FIG. 7 shows a construction scheme for plasmids, pChi796HMS1 andpChi750HMS1.

FIG. 8 shows a construction scheme for plasmids, pChi796LI1 andpChi750LI1.

FIG. 9 shows a construction scheme for plasmids, pChi796LM1 andpChi750LM1.

FIG. 10 shows a construction scheme for plasmids, pChi796LMS1 andpChi750LMS1.

FIG. 11 shows a construction scheme for plasmids, pChi796H107 andpChi750H107.

FIG. 12 shows a construction scheme for plasmids, pChi796HL1 andpChi750HL1.

FIG. 13 shows a construction scheme for a plasmid, pChi603HM1.

FIG. 14 shows a construction scheme for a plasmid, pChi603HMS1.

FIG. 15 shows a construction scheme for a plasmid, pChi603LI1.

FIG. 16 shows a construction scheme for a plasmid, pChi603LM1.

FIG. 17 shows a construction scheme for a plasmid, pChi603LMS1.

FIG. 18A and 18B show the electrophoretic patterns in SDS-PAGE (using4-15% gradient gels) of purified chimeric human anti-GM₂ antibodies,KM966 and KM967. The patterns obtained under reducing conditions areshown on the left side and those obtained under nonreducing conditionson the right side. From the left, the lanes include low molecular weightmarkers, KM967 and KM966 (reducing conditions), and KM967 and KM966(nonreducing conditions).

FIGS. 19A and 19B show the electrophoretic patterns in SDS-PAGE (using4-15% gradient gels) of a purified chimeric human anti-GM₂ antibody,KM968. The pattern obtained under reducing conditions is shown on theleft side and that obtained under nonreducing conditions on the rightside. From the left, the lanes include high molecular weight markers,low molecular weight markers, a standard human IgG, KM968 (reducingconditions), the same low molecular weight markers, the standard humanIgG, and KM968 (nonreducing conditions).

FIGS. 20A and 20B graphically show the CDC (complement dependentcytotoxicity) activities of KM966 against the human lung small cellcarcinoma cell lines SBC-3 and LU-135. The ordinate indicates thecytotoxic-activity and the abscissa the concentration of the antibodyadded. The solid bars indicate the CDC activities of KM-696 and theshaded bars the CDC activities of KM966.

FIGS. 20A and 21B graphically show the CDC activities of KM966 againstthe human lung squamous cell carcinoma cell line PC-10 and human lungadenocarcinoma cell line RERF-LC-MS. The ordinate indicates thecytotoxicity and the abscissa the concentration of the antibody added.The solid bars indicate the CDC activities of KM-696 and the shaded barsthe CDC activities of KM966.

FIGS. 22A and 22B graphically show the CDC activities of KM966 againstthe human lung large cell carcinoma cell line PC-13 and humanneuroblastoma cell line NAGAI. The ordinate indicates the cytotoxicactivity and the abscissa the concentration of the antibody added. Thesolid bars indicate the CDC activities of KM-696 and the shaded bars theCDC activities of KM966.

FIGS. 23A and 23B graphically show the CDC activities of KM966 againstthe human neuroblastoma cell line GOTO and human brain tumor cell lineA172. The ordinate indicates the cytotoxic activity and the abscissa theconcentration of the antibody added. The solid bars indicate the CDCactivities of KM696 and the shaded bars the CDC activities of KM966.

FIGS. 24A and 24B graphically show the ADCC (antibody dependent cellmediated cytotoxicity) activities of KM966 against the human lung smallcell carcinoma cell lines SBC-3 and LU-135. The ordinate indicates thecytotoxic activity and the abscissa the concentration of the antibodyadded. The solid bars indicate the ADCC activities of KM-696 and theshaded bars the ADCC activities of KM966.

FIGS. 25A and 25B graphically show the ADCC activities of KM966 againstthe human lung squamous carcinoma cell line PC-10 and human lungadenocarcinoma cell line RERF-LC-MS. The ordinate indicates thecytotoxic activity and the abscissa the concentration of the antibodyadded. The solid bars indicate the ADCC activities of KM-696 and theshaded bars the ADCC activities of KM966.

FIGS. 26A and 26B graphically show the ADCC activities of KM966 againstthe human lung large cell carcinoma cell line PC-13 and humanneuroblastoma cell line NAGAI. The ordinate indicates the cytotoxicityand the abscissa the concentration of the antibody added. The solid barsindicate the ADCC activities of KM-696 and the shaded bars the ADCCactivities of KM966.

FIGS. 27A and 27B graphically show the ADCC activities of KM966 againstthe human neuroblastoma cell line GOTO and human brain tumor cell lineA172. The ordinate indicates the cytotoxicity and the abscissa theconcentration of the antibody added. The solid bars indicate the ADCCactivities of KM-696 and the shaded bars the ADCC activities of KM966.

FIG. 28 shows a restriction enzyme cleavage map of a 9.3 kb XbaIfragment of the KM50 cell chromosomal DNA.

FIG. 29 shows a construction scheme for a plasmid, pKMB11.

FIG. 30 shows a construction scheme for a plasmid, pKMD6.

FIG. 31 shows a construction scheme for a plasmid, pEPKMA1.

FIG. 32 shows a construction scheme for a plasmid, pEPKMB1.

FIG. 33 shows a construction scheme for a plasmid, pAGE501.

FIG. 34 shows a construction scheme for a plasmid, pAGE109.

FIG. 35 shows a construction scheme for a plasmid, pAGE502.

FIG. 36 shows a construction scheme for a plasmid, pAGE503.

FIG. 37 shows a construction scheme for a plasmid, pSEd1.

FIG. 38 shows a construction scheme for a plasmid, pSE1D2.

FIG. 39 shows a construction scheme for a plasmid, pIG1SE1d2.

FIG. 40 shows a construction scheme for a plasmid, pIG1SE1d3.

FIG. 41 shows a construction scheme for a plasmid, pIG1SE1d4.

FIG. 42 shows a construction scheme for a plasmid, pPMOL2.

FIG. 43 shows a construction scheme for a plasmid, pPMOL3.

FIG. 44 shows a construction scheme for a plasmid, pchCKA7.

FIG. 45 shows a construction scheme for a plasmid, pchCKB1.

FIG. 46 shows a construction scheme for a plasmid, pckCKC1.

FIG. 47 shows a construction scheme for a plasmid, pChiIgHB2.

FIG. 48 shows a construction scheme for a plasmid, pChiIgLA1.

FIG. 49 shows a construction scheme for plasmids, pKM641HA3 andpKM641LA2.

FIG. 50 shows a construction scheme for a plasmid, pChi641HA1.

FIG. 51 shows a construction scheme for a plasmid, pKM641HE1.

FIG. 52 shows a construction scheme for a plasmid, pKM641HF1.

FIG. 53 shows a construction scheme for a plasmid, pChi641HA1.

FIG. 54 shows a construction scheme for a plasmid, pChi641HAM1.

FIG. 55 shows a construction scheme for a DNA, hKM796H.

FIG. 56 shows a construction scheme for a DNA, hKM796L.

FIG. 57 shows a construction scheme for a plasmid, phKM796HM1.

FIG. 58 shows a construction scheme for a plasmid, phKM796HMS1.

FIG. 59 shows a construction scheme for a plasmid, phKM796LI1.

FIG. 60 shows a construction scheme for a plasmid, phKM796LM1.

FIG. 61 shows a construction scheme for a plasmid, PhKM796LMS1.

FIG. 62 shows a construction scheme for a plasmid, phKM796H107.

FIG. 63 shows a construction scheme for a plasmid, phKM796HL1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to humanized antibodies specific for theganglioside GM₂. The antibodies can be of any of the immunoglobulin (Ig)classes, it is preferable, however, that the antibodies be of the IgGtype. The term "humanized antibody", as used herein, includes within itsmeaning, chimeric human antibody and CDR-grafted antibodies. Chimerichuman antibodies of the invention include the V_(H) and V_(L) of anantibody of an animal other than a human and the C_(H) and C_(L) of ahuman antibody. The CDR-transplanted antibodies of the invention resultfrom the replacement of CDRs of the V_(H) and V_(L) of a human antibodywith those of the V_(H) and V_(L), respectively, of an antibody of ananimal other than a human.

An example of a chimeric human antibody of the invention is an antibodythe V_(H) of which contains an amino acid sequence segment as defined bySEQ ID NO:1, including the 1st to 120th amino acids of that sequence,and the V_(L) of which contains an amino acid sequence segment asdefined by SEQ ID NO:2, including the 1st to 107th amino acids of thatsequence.

An example of a CDR-transplanted antibody of the invention is anantibody the V_(H) CDRs of which have the amino acid sequences definedby SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 and the V_(L) CDRs of whichhave the amino acid sequences defined by SEQ ID NO:9, SEQ ID NO:10 andSEQ ID NO:11.

The chimeric human antibodies of the invention can be produced in thefollowing manner:

(1) Preparation of cDNAs coding for the V_(H) and V_(L) of an antibodyof nonhuman animal

cDNAs coding for the V_(H) and V_(L) of an antibody of a nonhumananimal, for example a mouse anti-GM₂ monoclonal antibody, can beprepared as follows.

mRNAs can be extracted from hybridomas producing the mouse anti-GM₂monoclonal antibody, for example hybridomas producing the mouse anti-GM₂monoclonal antibody KM796, and cDNAs reverse transcribed therefrom.Using the cDNAs, a library can be constructed using phage or plasmidvectors. The recombinant phage or recombinant plasmid containing thecDNA coding for the V_(H), and the recombinant phage or recombinantplasmid containing the cDNA coding for the V_(L) can be isolated fromthe library using a constant region portion or a variable region portionof an antibody of a nonhuman animal, for example a mouse antibody, as aprobe. The base sequences of the V_(g) -encoding cDNA and V_(L)-encoding cDNA in the recombinant phage or recombinant plasmid can thenbe determined. Examples of the nonhuman animals include mice, rats,hamsters and monkeys.

(2) Construction of a vector for chimeric human antibody expression

Expression of chimeric human antibody H chain and L chains can beeffected using expression vectors suitable for use in animal cells,inserted into which are the cDNAs coding for the human C_(H) and C_(L).Any expression vector suitable for use in animal cells can be used,provided that it allows integration and expression of the human antibodyconstant region-encoding cDNAs. Examples include pAGE107 Cytotechnology,3, 133 (1990)!, pAGE103 J. Biochem., 101, 1307 (1987)!, pHSG274 Gene,27, 223 (1984)!, PKCR Proc. Natl. Acad. Sci. U.S.A., 78, 1527 (1981)!and pSG1βd2-4 Cytotechnology, 4, 173 (1990)!, among others. Examples ofpromoters and enhancers suitable for use in such expression vectorsinclude the SV40 early promoter and enhancer J. Biochem., 101, 1307(1987)!, the Moloney mouse leukemia virus LTR (long terminal repeat)promoter and enhancer Biochem. Biophys. Res. Commun., 149, 960 (1987)!and the immunoglobulin H chain promoter Cell, 41, 479 (1985)! andenhancer Cell, 33, 717 (1983)!. The promoters and enhancers are locatedin the expression vector in operable linkage with the coding sequences.

(3) Construction of a chimeric human antibody expression vector

The vector for chimeric human antibody H chain and L chain expression,as obtained in (2), is provided with a cloning site upstream of thehuman constant region, for insertion of a cDNA coding for the variableregion of an antibody of a nonhuman animal. Insertion, at this cloningsite, of the cDNA coding for the variable region of a nonhuman animalantibody, using a synthetic DNA comprising a 5' terminal base sequenceof the human antibody constant region and a 3' terminal base sequence ofthe variable region of the nonhuman animal antibody and havingrestriction enzyme sites on both ends, gives a chimeric human antibodyexpression vector with the cDNA coding for the human antibody constantregion and the cDNA coding for the variable region of the nonhumananimal antibody joinedly inserted therein via the synthetic DNA forproducing appropriate restriction enzyme sites. The synthetic DNA can besynthesized using a DNA synthesizer based on the 5' terminal basesequence of the human antibody constant region and the base sequence ofsaid 3' terminal base sequence of the nonhuman animal antibody variableregion.

(4) Construction of a chimeric human antibody H chain expression vector

A vector for chimeric human antibody H chain expression is constructed,for example, by excising that portion of the human antibody C_(H)-encoding cDNA which covers from the ApaI site near the 5' terminus tothe 3' terminus and inserting that portion into an expression vectorsuitable for use in animal cells. This vector for chimeric humanantibody H chain expression is provided with a cloning site forinsertion of a cDNA coding for a nonhuman animal V_(H). cDNA coding forthe nonhuman animal V_(H), excised using an appropriate restrictionenzyme, is inserted into the vector at the cloning site using asynthetic DNA comprising that portion of the human antibody C_(H) genewhich covers from the 5' terminus to the ApaI site and the base sequenceof a 3' terminal portion of the nonhuman animal antibody V_(H) gene andhaving restriction enzyme sites on both ends, to give a chimeric humanantibody H chain expression vector which allows no change in the aminoacid sequence of V_(H) upon expression thereof and has appropriaterestriction enzyme sites.

(5) Construction of a chimeric human antibody L chain expression vector

A vector for chimeric human antibody L chain expression is constructed,for example by introducing an EcoRV site into the human antibody C_(L)-encoding cDNA in the vicinity of the 5' terminus by mutagenesis,excising that portion which covers from the EcoRV site to the 3'terminus and inserting that portion into a plasmid, such as the plasmidpIg1SE1d4. This vector for chimeric human antibody L chain expression isprovided with a cloning site for insertion of the cDNA coding fornonhuman animal V_(L). The nonhuman animal antibody V_(L) -encodingcDNA, excised with an appropriate restriction enzyme, is inserted intothe vector at the cloning site using a synthetic DNA comprising thatportion of the human antibody C_(L) gene which covers from the 5'terminus to the EcoRV site and the base sequence of a 3' terminalportion of the nonhuman animal antibody V_(L) gene and havingrestriction enzyme sites on both ends, to give a chimeric human antibodyL chain expression vector which allows no change in the amino acidsequence of V_(L) upon expression thereof.

(6) Introduction of the chimeric human antibody expression vectors intohost cells

Introduction of the chimeric human antibody H chain expression vectorand chimeric human antibody L chain expression vector into host cellsgives a transformant producing the chimeric human antibody. Inintroducing the vectors into host cells, a splicing signal may beintroduced into the chimeric human antibody H chain and L chainexpression vectors for mRNA stabilization Cell, 17, 737 (1979)!.

The chimeric human antibody H chain and L chain vectors can beintroduced into host cells, for example, simultaneously byelectroporation JP-A-2-257891 (the term "JP-A" used herein means anunexamined published Japanese patent application.); Cytotechnology, 3,133 (1990)!. In addition, an expression vector containing genes codingfor both the chimeric human antibody H chain and L chain tandemexpression vector! can be introduced into host cells BIO/TECHNOLOGY, 9,64 (1991)!. The use of a tandem expression vector is preferred since ahigher level of chimeric human antibody expression can be attainedthereby, with approximately equal H chain and L chain expression levels.

An example of a method of producing the CDR-transplanted antibodies ofthe invention is described as follows.

First, a CDR-transplanted antibody expression vector can be constructedby the method of Winter et al. Nature, 332, 323 (1988)! as follows.

Three synthetic DNAs are constructed designed so as to comprise thecDNAs coding for three CDR peptides of the V.sub. H of a nonhuman animalantibody, for example, peptides having the amino acid sequences definedby SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, with DNAs coding for aminoacid sequences comprising of several amino acids from both ends of thecorresponding CDRs of the V_(H) of a human antibody being located at therespective both ends of the cDNAs, DNA synthesis is carried out with aplasmid containing the human antibody V_(H) gene as a template. Anexample of the human antibody V_(H) gene-containing plasmid is the M13plasmid containing a human antibody NEW gene-derived sequence J. Biol.Chem., 253, 585 (1978); Nature, 332, 323 (1988)!.

The DNA obtained is inserted into the vector for chimeric human antibodyH chain expression in the same manner as in the construction of thechimeric human antibody expression vector mentioned above to give aCDR-transplanted antibody H chain expression vector.

Similarly, using, as primers, three synthetic DNAs designed to comprisethe cDNAs coding for three CDR peptides of the V_(L) of a nonhumananimal antibody, for example, the peptides having the amino acidsequences defined by SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, withDNAs coding for amino acid sequences comprising several amino acids fromboth ends of the corresponding CDRs of the human antibody V_(L) beinglocated at the respective both ends of said cDNAs, DNA synthesis iscarried out with a human antibody V_(L) gene-containing plasmid as atemplate. An example of the human antibody V_(L) gene-containing plasmidis the M13 plasmid containing a human myeloma protein (Bence-Jonesprotein) REI gene-derived sequence Eur. J. Biochem., 45, 513 (1974);Nature, 332, 323 (1988)!.

By inserting the DNA obtained into a vector for chimeric human L chainexpression in the same manner as described in respect of theconstruction of the chimeric human antibody expression vector, aCDR-transplanted antibody L chain expression vector can be constructed.

It is also possible to construct the CDR-transplanted antibody H chainand L chain expression vectors by synthesizing DNAs coding for thepeptides having amino acid sequences resulting from replacement of thethree CDRs each of the H chain and L chain of a human antibody with thecorresponding CDRs of the H chain and L chain of a nonhuman animalantibody and then inserting the DNAs into a vector for chimeric humanantibody H chain or L chain expression in the same manner as describedin respect of the construction of the chimeric human antibody expressionvector mentioned above.

The CDR-transplanted antibody expression vector can be introduced intohost cells in the same manner as the chimeric human antibody expressionvector to give a transformant producing the CDR-transplanted antibody.

The host cells suited for the introduction thereinto of the chimerichuman antibody or CDR-transplanted antibody expression vector may be anyhost cells provided that the chimeric human antibody or CDR-transplantedantibody can be expressed therein. Examples include mouse SP2/0-Ag14cells (ATCC CRL1581; hereinafter, "SP2/0 cells"), mouse P3x63-Ag8.653cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductasegene (hereinafter, "dhfr") Urlaub et al.: Proc. Natl. Acad. Sci. U.S.A.,77, 4216 (1980)! and rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662;hereinafter, "YB2/0 cells"), with YB2/0 cells being preferred.

The transformants producing the chimeric human antibody orCDR-transplanted antibody are selected by the method disclosed inJP-A-2-257891 using PRMI1640 medium containing G418 and fetal calfserum. A particular example of the chimeric human antibody-producingtransformant is the transformant KM966 producing a chimeric humanantibody that reacts with the ganglioside GM₂. --KM966 has beendeposited with the Fermentation Research Institute, Agency of IndustrialScience and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki305 JAPAN, as of Jul. 15, 1992 under the deposit number FERM BP-3931.

When the transformant obtained is cultivated in a medium, the chimerichuman antibody or CDR-transplanted antibody can be produced andaccumulated in the culture fluid. The activity of the chimeric humanantibody or CDR-transplanted antibody in the medium can be determined byan enzyme-linked immunosorbent assay (ELISA; E. Harlow et al. (ed.):Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).The antibody productivity of the transformant can be increased byutilizing a dhfr amplification system as disclosed in JP-A-2-257891.

The chimeric human antibody and CDR-transplanted antibody can bepurified from the culture supernatants obtained as mentioned above usinga protein A column (E. Harlow et al. (ed.): Antibodies--A LaboratoryManual, Cold Spring Harbor Laboratory, 1988). As noted above, thechimeric human antibody KM966, which reacts with the ganglioside GM₂, isa specific example of the thus-obtained chimeric human antibodies andCDR-transplanted antibodies.

The reactivity of the chimeric human antibody or CDR-transplantedantibody of the invention can be checked by ELISA. The molecuar weightof the purified antibody H chain or L chain or whole antibody moleculecan be determined by polyacrylamide gel electrophoresis (SDS-PAGE) orWestern blotting (E. Harlow et al. (ed.): Antibodies--A LaboratoryManual, Cold Spring Harbor Laboratory, 1988).

The binding activity of the chimeric human antibody or CDR-transplantedantibody that reacts with the ganglioside GM₂ of cultured cancer cellscan be measured, for example, by the fluorescent antibody technique orby ELISA. The complement dependent cytotoxic activity (CDC activity) andantibody dependent cell mediated cytotoxic activity (ADCC activity) ofthe chimeric human antibody or CDR-transplanted antibody are measured bythe methods described in the monograph "Men-ekigaku Jikken Nyumon (AManual of Experiments in Immunology)" (Matsuhashi et al., published byGakkai Shuppan Center, 1981).

The humanized antibodies of the invention specifically bind to humancancer cells and exhibit CDC activity and ADCC activity against humancancer cells and therefore are useful in the treatment of human cancers,among others.

The humanized antibodies according to the present invention can be usedalone as an anticancer agent. They may be formulated into an anticancercomposition together with at least one pharmaceutically acceptablecarrier. For instance, the humanized antibodies are dissolved inphysiological saline, an aqueous solution of glucose, lactose ormannitol and the like. The powder of the humanized antibodies forinjection can be prepared by lyophilizing the humanized antibodies inaccordance with the conventional method and mixing the lyophilizedproducts with sodium chloride. The anticancer composition may furthercontain additives conventionally used well known in the art of medicalpreparation, for example, pharmaceutically acceptable salts.

The humanized antibodies according to the present invention can beadministered in the form of the above-described anticancer compositionto mammals including human in a dose of 0.2 to 20 mg/kg/day. The dosemay vary depending on the age, condition, etc. of patients. Theadministration of the anticancer composition can be effected byintravenous injection once a day (single administration or consecutiveadministration) or intermittently one to three times a week or onceevery two to three weeks

The anticancer composition is expected to be useful for treating cancersuch as melanoma, neuroblastoma and glioma.

The following Examples and Reference Examples are further illustrativeof the present invention, but are not to be construed to limit the scopeof the present invention.

EXAMPLE 1

Production of chimeric human anti-GM₂ antibodies

1. Isolation of mRNAs from hybridoma cells producing the mouse anti-GM₂monoclonal antibody KM-796 or KM-750 and from hybridoma cells producingthe rat anti-GM₂ monoclonal antibody KM-603

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitorogen and following the description of the manualattached to the kit, mRNAs were isolated from 1×10⁸ cells each of themouse anti-GM₂ monoclonal antibody KM-796-producing hybridoma cell line(FERM BP-3340), the mouse anti-GM₂ monoclonal antibody KM-750-producinghybridoma cell line (FERM BP-3339) and the rat anti-GM₂ monoclonalantibody KM-603-producing hybridoma cell line (FERM BP-2636).

2. Construction of monoclonal antibody KM-796 and KM-750 H chain and Lchain cDNA libraries

Using cDNA Synthesis Kit (product number 27-9260-01) manufactured byPharmacia and following the manual attached to the kit, cDNA having theEcoRI adapter on both ends was synthesized from 5 μg each of the KM-796-and KM-750-derived mRNAs obtained as described in Paragraph 1 above.About 6 μg of each cDNA product obtained was dissolved in 10 μl ofsterilized water and fractionated by agarose gel electrophoresis, and acDNA fragment (about 1.8 kb) corresponding to the IgG antibody H chainand a cDNA fragment (about 1.0 kb) corresponding to the L chain wererecovered (about 0.1 μg each). Then, 0.1 μg of each cDNA fragment ofabout 1.8 kb and 0.1 μg of each cDNA fragment of about 1.0 kb wererespectively dissolved in 11.5 μl of T4 ligase buffer, together with 1μg of the Lambda ZAPII vector (cleaved with EcoRI and then treated withcalf intestine alkaline phosphatase; product of Stratagene). Afteraddition of 175 units of T4 DNA ligase, each solution was incubated at12° C. for 24 hours and then at room temperature for 2 hours. A 4-μlportion of each reaction mixture was subjected to packaging into thelambda phage in the conventional manner Maniatis et al. (ed.): MolecularCloning, 2.95 Cold Spring Harbor Laboratory, 1989! using Giga Pak Gold(Stratagene), followed by transfection, in the conventional mannerManiatis et al. (ed.): Molecular Cloning, 2.95-107, Cold Spring HarborLaboratory, 1989! of the Escherichia coli strain XL1-Blue Biotechniques,5, 376 (1987)! attached to Giga Pak Gold, to give about 4,000 phageclones each as a KM-796 or KM-750 H chain or L chain cDNA library. Thenthe phage clones of each library were fixed on a nitrocellulose filterin the conventional manner Maniatis et al. (ed.): Molecular Cloning,2.112, Cold Spring Harbor Laboratory, 1989!.

3. Construction of KM-603 H chain and L chain cDNA libraries

Using 5 μg of the KM-603 mRNA obtained as mentioned above in Paragraph 1and cDNA Synthesis Kit (product number 27-9260-01) manufactured byPharmacia, cDNA having the EcoRI adapter on both ends was synthesized.About 6 μg of the cDNA produced was dissolved in 10 μl of sterilizedwater and fractionated by agarose gel electrophoresis. A cDNA fragment(about 2.2 kb) corresponding to the IgG antibody H chain and a cDNAfragment (about 1.0 kb) corresponding to the L chain were recovered(about 0.1 μg each). Then 0.1 μg of the cDNA fragment of about 2.2 kband 0.1 μg of the cDNA fragment of about 1.0 kb were respectivelydissolved in 11.5 μl of T4 ligase buffer, together with 1 μg of theLambda ZAPII vector (cleaved with EcoRI and then treated with calfintestine alkaline phosphatase; product of Stratagene) and, afteraddition of 175 units of T4 DNA ligase, the resultant solution wasincubated at 12° C. for 24 hours and then at room temperature for 2hours. A 4-μl portion of each reaction mixture was subjected topackaging into the lambda phage in the conventional manner Maniatis etal. (ed.): Molecular Cloning, 2.95, Cold Spring Harbor Laboratory, 1989!using Giga Pak Gold (Stratagene), followed by transfection, in theconventional manner Maniatis et al. (ed.): Molecular Cloning, 2.95-107,Cold Spring Harbor Laboratory, 1989!, of the Escherichia coli strainXL-Blue attached to Giga Pak Gold, whereby about 10,000 phage cloneswere obtained each as a KM-603 H chain or L chain cDNA library. Then,the phage clones of each library were fixed on a nitrocellulose filterin the conventional manner Maniatis et al. (ed.): Molecular Cloning,2.112, Cold Spring Harbor Laboratory, 1989!.

4. Cloning of the KM-796 and KM-750 H chain and L chain cDNAs

From among the KM-796 and KM-750 H chain cDNA libraries and L chain cDNAlibraries constructed as described above in Paragraph 2, phage clonesfirmly bound at 65° C. to a probe prepared by labeling a mouseimmunoglobulin constant region cDNA for the H chain, the BamHI-XhoIfragment of the mouse Cγ3 cDNA (Wels et al: EMBO J., 3, 2041-2046,1984); for the L chain, the HpaI-XhoI fragment of the mouse Cκ cDNA(Hieter et at.: Cell, 22, 197-207, 1980)! with ³² P were recovered inthe conventional manner Maniatis et al.: Molecular Cloning, 2.108, ColdSpring Harbor Laboratory, 1989!. Then, using a ZAP-cDNA Synthesis Kit(cDNA synthesis kit; product number sc200400) manufactured byStratagene, phage clones were converted into pBluescript plasmids, and aKM-796 H chain cDNA-containing recombinant plasmid (pKM796H1) and aKM-796 L chain cDNA-containing recombinant plasmid (pKM796L1) (FIG. 1)as well as a KM-750 H chain cDNA-containing recombinant plasmid(pKM750H1) and a KM-750 L chain cDNA-containing recombinant plasmid(pKM750L1) (FIG. 2) were obtained. Cleavage of pKM796H1, pKM750H1,pKM796L1 and pKM750L1 with EcoRI revealed that a cDNA fragment of about1.8 kb had been inserted into pKM796H1 and pKM750H1 and a cDNA fragmentof about 0.9 kb into pKM796L1 and pKM750L1.

5. Cloning of KM-603 H chain and L chain cDNAs

Phage clones firmly bound at 65° C. to a probe prepared by labeling amouse immunoglobulin constant region chromosomal gene mouse Cμgene-containing SmaI-KpnI fragment of about 11.5 kb (Kataoka et al.:Proc. Natl. Acad. Sci. U.S.A., 77, 919-923, 1980) and mouse Cκgene-containing HindIII-BamHI fragment of about 3 kb (Sakano et al.:Nature, 280, 288, 1979)! with³² P were isolated from the KM-603 H chaincDNA library and L chain cDNA library constructed as mentioned above inParagraph 3 in the conventional manner Maniatis et al. (ed.): MolecularCloning, 2.108, Cold Spring Harbor Laboratory, 1989!. Then, usingZAP-cDNA Synthesis kit (product number sc200400) manufactured byStratagene, the phage clones were converted to pBluescript plasmids anda KM-603 H chain cDNA-containing recombinant plasmid, pKM603H1, and aKM-603 L chain cDNA-containing recombinant plasmid, pKM603L1, wereobtained (FIG. 3). Cleavage of pKM603H1 and pKM603L1 revealed thatpKM603H1 contained a cDNA fragment of about 2.0 kb as inserted thereinand pKM603L1 a cDNA fragment of about 0.9 kb as inserted therein.

6. Base sequences of the variable regions in the H chain cDNA and Lchain cDNA

The base sequences of the variable regions in the H chain cDNA and Lchain cDNA obtained as mentioned above in Paragraphs 4 and 5 weredetermined by the dideoxy method Maniatis et al. (ed.): MolecularCloning, 13.42, Cold Spring Harbor Laboratory, 1989! using SequenaseVersion 2.0 DNA Sequencing Kit manufactured by United States BiochemicalCorporation. All the cDNA had a methionine codon, presumably theinitiation codon ATG, at the 5' terminus and were leadersequence-containing full-length cDNAs. Based on the base sequences ofthe respective cDNAs, the amino acid sequences of the H chain and Lchain of KM-796, KM-750 and KM-603 were deduced. The amino acid sequenceof the KM-796 H chain is shown in SEQ ID NO:1, that of the L chain ofKM-796 and KM-750 in SEQ ID NO:2, that of the KM-750 H chain in SEQ IDNO:3, that of the KM-603 H chain in SEQ ID NO:4 and that of the KM-603 Lchain in SEQ ID NO:5.

7. Construction of KM-796- and KM-750-derived chimeric human antibody Hchain and L chain expression vectors

(1) Construction of a vector, pAGE147, carrying the Moloney mouseleukemia virus terminal repeat promoter/enhancer

The plasmid pPMOL1 (2 μg), described in JP-A-1-63394, was dissolved in30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mMmagnesium chloride and 6 mM 2-mercaptoethanol, 20 units of SmaI wasadded, and digestion was carried out at 30° C. for 3 hours. Then, sodiumchloride was added to a concentration of 50 mM, 20 units of ClaI wasadded, and digestion was conducted at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis, and a DNA fragment(about 0.6 kb) containing the Moloney mouse leukemia virus terminalrepeat promoter/enhancer was recovered.

Then, the following two synthetic DNAs were synthesized using anautomatic DNA synthesizer (model 380A manufactured by Applied BiosystemsCo., Ltd.). ##STR1##

The thus-obtained synthetic DNAs (25 picomoles each) were dissolved in10 μl of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT (dithiothreitol), 0.1 mM EDTA and 0.5 mMadenosine triphosphate (hereinafter, "ATP"), 5 units of T4 DNA kinasewas added, and 5'-phosphorylation was carried out at 37° C. for 30minutes.

The plasmid pPMOL1-derived ClaI-SmaII fragment (0.6 kb, 0.05 μg) and two5'-phosphorylated synthetic DNAs (1 picomole each), obtained asdescribed above, and a HindIII linker (5'-pCAAGCTTG-3'; Takara Shuzo) (1picomole) were dissolved in 30 μl of 66 mM Tris-hydrochloride buffer (pH7.5) containing 6.6 mM magnesium chloride, 10 mM DTT and 0.1 mM ATP, 200units of T4 DNA ligase (Takara Shuzo; hereinafter the same shall apply)were added, and ligation was carried out at 12° C. for 16 hours. Theresultant DNA fragment was recovered by ethanol precipitation anddissolved in 20 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added,and digestion was carried out at 37° C. for 2 hours. The reaction wasterminated by phenol-chloroform extraction, and the DNA fragment wasrecovered by ethanol precipitation.

Separately, 1 μg of the plasmid pAGE107 Cytotechnology, 3, 133 (1990)!was dissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride, 100 mM sodium chloride and 6 mM2-mercaptoethanol, 10 units of HindIII and 10 units of XhoI were added,and digestion was carried out at 37° C. for 2 hours. The reactionmixture was subjected to agarose gel electrophoresis, and a DNA fragment(about 6.0 kb) containing the G418 resistance gene and ampicillin(hereinafter, "Ap") resistance gene was recovered.

The plasmid pAGE107-derived HindIII-XhoI fragment (6.0 kb, 0.3 μg) andplasmid pPMOL1-derived HindIII-XhoI fragment (0.63 kb, 0.01 μg) obtainedas mentioned above were dissolved in 20 μl of 66 mM Tris-hydrochloridebuffer (pH 7.5) containing 6.6 mM magnesium chloride, 10 mM DTT and 0.1mM ATP, 200 units of T4 DNA ligase were added, and ligation was carriedout at 12° C. for 16 hours. The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101, and the plasmid pAGE147shown in FIG. 4 was obtained.

(2) Construction of a vector, pAGE148, carrying the β-globulin 3'splicing signal (SP)

For introducing the β-globulin 3' splicing signal into the chimerichuman antibody expression vector at a site downstream from the antibodyconstant region gene, a vector (pAGE148), was constructed as follows,which contained the β-globulin 3' splicing signal and the same genes asthose in the chimeric human antibody expression vector (except for thehuman antibody constant region gene).

Two μg of pSE1UK1SEd1-3, described in JP-A-2-257851, were added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT. After addition of 10 unitsof HindIII, digestion was carried out at 37° C. for 4 hours. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation. The precipitate was dissolved in 20 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment were added, and the 5' cohesive ends produced byHindIII digestion were rendered blunt by incubation at 22° C. for 30minutes. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT and 10 units of KpnI were added, and digestion was effectedat 37° C. for 4 hours. The reaction mixture was subjected tophenolchloroform extraction and then to ethanol precipitation, 30 μl of50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT and 10 units of XhoI wereadded, and digestion was carried out at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and two DNAfragments, about 6.67 kb and about 1.98 kb in size, were recovered(about 0.2 μg each).

Then, 2 μl of pAGE147 obtained in (1) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT, 10 units of LpnI was added, and digestion was effected at37° C. for 4 hours. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, 30 μl of50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT and 10 units of XhoI wereadded, and digestion was carried out at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 0.2 μgof a DNA fragment of about 0.66 kb was recovered.

Then, 0.1 μg of the XhoI-HindIII fragment (about 6.67 kb) ofpSE1UK1SEd1-3, as obtained above, 0.1 μg of the KpnI-HindIII fragment(about 1.98 kb), obtained above, and 0.1 μg of the XhoI-KpnI fragment(about 0.66 kb) of pAGE147, as obtained above, were dissolved in a totalof 20 μl of T4 ligase buffer. Three hundred fifty units of T4 ligasewere added to the solution, and ligation was carried out at 4° C. for 24hours. The thus-obtained-recombinant plasmid DNA was used to transformEscherichia coli HB101, and the plasmid pAGE148 shown in FIG. 5 wasobtained.

(3) Construction of KM-796- and KM-750-derived chimeric human antibody Hchain expression vectors

First, the cDNA coding for the antibody variable region in the plasmidpKM796H1 or pKM750H1 was excised by cleavage at the 5'-terminal EcoRIsite and the MaeIII site near the 3' end of said cDNA and joined,together with a synthetic DNA having the base sequence shown in SEQ IDNO:12, to the chimeric human antibody H chain expression vectorpChi641HAM1, as follows (FIG. 6).

Three μg of pKM796H1 or pKM750H1, obtained in Paragraph 4, were added to30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT. Further, 10units of EcoRI and 10 units of MaeIII were added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about0.43 kb was recovered. Then, 3 μg of pChi641HAM1, obtained in ReferenceExample 2, was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10 units of EcoRIand 10 units of ApaI were also added, and digestion was carried out at37° C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.0 μg of a DNA fragment of about 9.0 kb wasrecovered. Then, 0.1 μg of the EcoRI-MaeIII fragment (about 0.43 kb) ofpKM796H1 or pKM750H1, as obtained above, 0.1 μg of the EcoRI-ApaIfragment (about 9.0 kb) of pChi641HAM1, as obtained above, and 0.3 μg ofa synthetic DNA having the base sequence shown in SEQ ID NO:12 weredissolved in a total of 20 μl of T4 ligase buffer, 350 units of T4ligase was further added to the solution, and ligation was carried outat 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101. In this way, the plasmidspChi796HM1 and pChi750HM1, shown in FIG. 6, were obtained.

Then, the β-globulin 3' splicing signal was introduced into the plasmidspChi796HM1 and pChi750HM1 by the method described below to constructKM796- and KM-750-derived chimeric human antibody H chain expressionvectors (FIG. 7).

Three μg of pChi796HM1 or pChi750HM1 were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% bovine serum albumin(hereinafter, "BSA"). Ten units of XhoI and 10 units of KpnI were alsoadded, and digestion was carried out at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 0.3 μgof a DNA fragment of about 3.4 kb was recovered. Then, 3 μg of pAGE148obtained in (2) was added to 30 μl of 33 mM Tris-acetate buffer (pH 7.9)containing 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 mM DTTand 0.01% BSA; 10 units of XhoI and 10 units of KpnI were further added,and digestion was effected at 37° C. for 4 hours. The reaction mixturewas fractionated by agarose gel electrophoresis and about 0.3 μg of aDNA fragment of about 8.7 kb was recovered. Then, 0.1 μg of theXhoI-KpnI fragment of pChi796HM1 or pKM750HM1.and00.1 μg of theXhoI-KpnI fragment of pAGE148 were dissolved in a total of 20 μl of T4ligase buffer, 350 units of T4 ligase was further added to the solution,and ligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101.The plasmids pChi796HMS1 and pChi750HMS1 shown in FIG. 7 were thusobtained.

(4) Construction of KM-796- and KM-750-derived chimeric human antibody Lchain expression vectors

First, the cDNA coding for the antibody variable region in the plasmidpKM796L1 or pKM750L1 was excised by cleavage at the 5'-terminal EcoRIsite and the Af1III site near the 3' end of said cDNA and joined,together with a synthetic DNA having the base sequence shown in SEQ IDNO:13, to the chimeric human antibody L chain expression vectorpChiIgLA1, as follows (FIG. 8).

Three μg of pKM796L1 or pKM750L1 were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10units of Af1III were added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.39 kb wasrecovered. Then, 3 μg of pChiIgLA1 obtained in Reference Example 1 wasadded to 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were further added, and digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 1 μg of a DNA fragment of about8.6 kb was recovered.

Then, 0.1 μg of the EcoRI-Af1III fragment of pKM796L1 or pKM750L1, asobtained above, 0.1 μg of the EcoRI-EcoRV fragment of pChiIgLA1, asobtained above, and 0.3 μg of a synthetic DNA, having the base sequenceshown in SEQ ID NO:13, were dissolved in a total of 20 μl of T4 ligasebuffer; 350 units of T4 ligase was further added to the solution, andligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101. Inthis way, the plasmids pChi796LI1 and pChi750LI1 shown in FIG. 8 wereobtained.

Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancerwas introduced into the plasmids pChi796LI1 and pChi750LI1 in thefollowing manner (FIG. 9).

Three μg of pChi796LI1 and pChi750LI1 were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Further, 10 units of EcoRI and 10units of XhoI were added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.2 kb wasrecovered Then, 3 μg of the chimric human antibody H chain expressionvector pChi641HAM1 obtained in Reference Example 2 was added to 30 l of50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10units of XhoI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.6 kb wasrecovered.

Then, 0.1 μg of the EcoRI-XhoI fragment of pChi796LI1 or pKM750LI1 asobtained above and 0.1 μg of the EcoRI-XhoI fragment of pChi641HAM1 asobtained above were dissolved in a total of 20 μl of T4 ligase buffer,350 units of T4 ligase was further added to the solution, and ligationwas carried out at 4° C. for 24 hours. The thus-obtained recombinantplasmid DNA was used to transform Escherichia coli HB101. In this way,the plasmids pChi796LM1 and pChi750LM1 shown in FIG. 9 were obtained.

Then, the β-globulin 3' splicing signal was introduced into the plasmidspChi796LM1 and pChi750LM1 in the manner mentioned below to constructKM-796- and KM-750-derived chimeric human antibody L chain expressionvectors (FIG. 10).

Three μg of pChi796LM1 or pChi750LM1 were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoIand 10 units of KpnI were added, and digestion was carried out at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 2.0 kb wasrecovered. Then, 3 μg of pAGE148 obtained in (2) was added to 30 μl of33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate,66 mM potassium acetate, 0.5 mM DTT and 0.01% BSA; 10 units of XhoI and10 units of KpnI were further added, and digestion was carried out at37° C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.7 kb wasrecovered. Then 0.1 μg of the XhoI-KpnI fragment of pChi796LM1 orpKM750LM1 as obtained above and 0.1 μg of the XhoI-KpnI fragment ofpAGE148 were dissolved in a total of 20 μl of T4 ligase buffer, 350units of T4 ligase was further added, and ligation was carried out at 4°C. for 24 hours. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101. In this way, the plasmids pChi796LMS1and pChi750LMS1 shown in FIG. 10 were obtained.

8. Construction of KM-796- and KM-750-derived chimeric human antibody Hchain and L chain tandem expression vectors

Tandem expression vectors containing the chimeric human antibody Hchain-encoding cDNA and L chain-encoding cDNA on one and the same vectorwere constructed (FIG. 11 and FIG. 12).

Three μg of pChi796HMS1 or pChi750HMS1, obtained in Paragraph 7, wereadded to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT. Further, 10units of MluI and 10 units of SalI were added, and digestion was carriedout at 37° C. for 4 hours. The reaction mixture was fractionated byarose gel electrophoresis. In each case, about 0.3 μg of a DNA fragmentof about 5.9 kb was recovered. Then, 2 μg of pAGE107 described in EP-A-0405 285 was dissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mMDTT; 10 units of MluI and 10 units of SalI were further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas fractionated by agarose gel electrophoresis and about 0.2 μg of aDNA fragment of about 3.55 kb was recovered. Then, 0.1 μg of theMluI-SalI fragment of pChi796HMS1 or pChi750HMS1 and 0.1 μg of theMluI-SalI fragment of pAGE107 were dissolved in a total of 20 μl of T4ligase buffer, 350 units of T4 ligase was added, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to give the plasmidpChi796H107 or pChi750H107 shown in FIG. 11.

Then, 3 μg of pChi796H107 or pChi750H107 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,50 mM sodium chloride and 1 mM DTT, 10 units of ClaI was further added,and digestion was carried out at 37° C. for 4 hours. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation. The precipitate was dissolved in 20 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the mixture was incubated at 22° C. for30 minutes for rendering the cohesive ends formed upon ClaI digestionblunt-ended. The reaction mixture was further subjected tophenol-chloroform extraction and then to ethanol precipitation. To theprecipitate were added 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT,and 10 units of MluI. Digestion was carried out at 37° C. for 4 hoursand the reaction mixture was fractionated by agarose gelelectrophoresis. In each case, about 0.3 μg of a DNA fragment of about7.5 kb was recovered. Then, 3 μg of pChi796LMS1 or pChi750LMS1 was addedto 30 μl of 20 mM Tris-hydrochloride buffer (pH 8.5) containing 10 mMmagnesium chloride, 100 mM potassium chloride and 1 mM DTT, 10 units ofXhoI was further added, and digestion was carried out at 37° C. for 4hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation. The precipitate wasdissolved in 20 μl of DNA polymerase I buffer, 5 units of Escherichiacoli-derived DNA polymerase I Klenow fragment was added, and the mixturewas incubated at 22° C. for 30 minutes for rendering the cohesive endsformed upon XhoI digestion blunt-ended. The reaction mixture was furthersubjected to phenol-chloroform extraction and then to ethanolprecipitation. To the precipitate was added 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,50 mM sodium chloride and 1 mM DTT as well as 10 units of MluI.Digestion was carried out at 37° C. for 4 hours and the reaction mixturewas fractionated by agarose gel electrophoresis. In each case, about 0.3μg of a DNA fragment of about 9.3 kb was recovered. Then, 0.1 μg of theMluI-ClaI fragment of pChi796H107 or pChi750H107, as obtained above, and0.1 μg of the MluI-XhoI fragment of pChi796LMS1 or pChi750LMS1, asobtained above, were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was further added, and ligation was carried outat 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101, and the plasmid pChi796HL1 orpChi750HL1 shown in FIG. 12 was obtained.

9. Construction of a KM-603-derived chimeric human antibody H chainexpression vector

First, the antibody variable region-encoding cDNA of the plasmidpKM603H1 was excised by cleavage at the 5'-terminal EcoRI site and theStyI site near the 3' end of said cDNA and joined to the chimeric humanantibody H chain expression vector pChi641HAM1 together with a syntheticDNA having the base sequence shown in SEQ ID NO:14 in the followingmanner (FIG. 13).

Three μg of pKM603H1 obtained in Paragraph 5 were added to 30 μl of 50mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, followed by furtheraddition of 10 units of EcoRI and 10 units of StyI. Digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 0.3 μg of a 0.4-kb DNA fragmentwas recovered. Then, 3 μg of pChi641HAM1, obtained in Reference Example2, was added to 30 μl of 10 mM Tris-hydrochloride (pH 7.5) containing 10mM magnesium chloride and 1 mM DTT, 10 units of EcoRI and 10 units ofApaI were further added, and digestion was effected at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.0 μg of a DNA fragment of about 9.0 kb wasrecovered. Then, 0.1 μg of the EcoRI-StyI fragment (about 0.4 kb) ofpKM603H1, as obtained above, and 0.1 μg of the EcoRI-ApaI fragment(about 9.0 kb) of pChi641HAM1, as obtained above, were dissolved,together with 0.3 μg of a synthetic DNA having the base sequence shownin SEQ ID NO:14, in a total of 20 μl of T4 ligase buffer; 350 units ofT4 ligase was added to the solution, and ligation was effected at 4° C.for 24 hours. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101 and the plasmid pChi603HM1 shown inFIG. 13 was obtained.

Then, a KM-603-derived chimeric human antibody H chain expression vectorwas constructed by introducing the β-globulin 3' splicing signal intothe plasmid pChi603HM1 in the following manner (FIG. 14).

Three μg of pChi603HM1 obtained above were added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA. Further, 10 units of XhoIand 10 units of KpnI were added, and digestion was carried out at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 3.3 kb wasrecovered.

Then, 3 μl of pAGE148 obtained in Paragraph 7 (2) was added to 30 μl of33 mM Tris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate,66 mM sodium acetate, 0.5 mM DTT and 0.01% BSA; 10 units of XhoI and 10units of KpnI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.7 kb wasrecovered. Then, 0.1 μg of the XhoI-KpnI fragment of pChi603HM1, asobtained above, and 0.1 μg of the XhoI-KpnI fragment of pAGE148, asobtained above, were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpChi603HMS1 shown in FIG. 14 was obtained.

10. Construction of a KM-603-derived chimeric human antibody L chainexpression vector

First, the antibody variable region cDNA in the plasmid pKM603L1 wasexcised by cleavage at the 5' terminal EcoRI site and the Af1III sitenear the 3' end and joined to the chimeric human antibody L chainexpression vector pChiIgLA1 together with a synthetic DNA having thebase sequence defined by SEQ ID NO:15 (FIG. 15).

Thus, 3 μg of pKM603L1 obtained in Paragraph 5 was added to 30 μl of 50mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10units of Af1III were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 0.4 kb wasrecovered. Then, 3 μg of pChiIgLA1 obtained in Reference Example 1 wasadded to 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were further added, and digestion wascarried out at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 1 μg of a DNA fragment of about8.6 kb was recovered. Then, 0.1 μg of the EcoRI-Af1III fragment ofpKM603L1, as obtained above, 0.1 μl of the EcoRI-EcoRV fragment ofpChiIgLA1, as obtained above, and 0.3 μg of a synthetic DNA, having thebase sequence defined by SEQ ID NO:15, were dissolved in a total of 20μl of T4 ligase buffer; 350 units of T4 ligase was added to thesolution, and ligation was carried out at 4° C. for 24 hours. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pChi603LI1 shown in FIG. 15 was obtained.

Then, the Moloney mouse leukemia virus terminal repeat promoter/enhancerwas introduced into the plasmid pChi603LI1 in the following manner (FIG.16).

Thus, 3 μg of pChi603LI1 obtained above was added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of EcoRI and 10 units ofXhoI were further added, and digestion was carried out at 37° C. for 4hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 8.3 kb wasrecovered. Then, 3 μg of pChi641HAM1 obtained in Reference Example 2 wasadded to 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of XhoI were further added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about0.6 kb was recovered. Then, 0.1 μg of the EcoRI-XhoI fragment ofpChi603LI1 as obtained above and 0.1 μg of the EcoRI-XhoI fragment ofpChi641HAM1 as obtained above were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Eschrichia coli HB101 togive the plasmid pChi603LM1 shown in FIG. 16.

A KM-603-derived chimeric human antibody L chain expression vector wasthen constructed by introducing the β-globulin 3' splicing signal intothe plasmid pChi603LM1, as follows (FIG. 17).

Thus, 3 μg of pChi603LM1 obtained above was added to 30 μl of 33 mMTris-acetate buffer (pH 7.9) containing 10 mM magnesium acetate, 66 mMpotassium acetate, 0.5 mM DTT and 0.01% BSA, 10 units of XhoI and 10units of KpnI were further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.3 μg of a DNA fragment of about 2.0 kb wasrecovered. Then, 3 μl of pAGE148 obtained in Paragraph 7 (2) was addedto 30 μl of 33 mM Tris-acetate buffer (pH 7.9) containing 10 mMmagnesium acetate, 66 mM potassium acetate, 0.5 mM DTT and 0.01% BSA, 10units of XhoI and 10 units of KpnI were further added, and digestion waseffected at 37° C. for 4 hours. The reaction mixture was fractionated byagarose gel electrophoresis and about 0.3 μg of a DNA fragment of about8.7 kb was recovered. Then, 0.1 μg of the XhoI-KpnI fragment ofpChi603LM1 as obtained above and 0.1 μg of the XhoI-KpnI fragment ofpAGE148, as obtained above, were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 ligase was added to the solution, andligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 togive the plasmid pChi603LMS1 shown in FIG. 17.

11. Expression of the KM-796- and KM-750-derived chimeric human anti-GM₂antibody in YB2/0 cells

The plasmids were introduced into YB2/0 cells by the electroporationmethod of Miyaji et al. Cytotechnbology, 3, 133-140 (1990)!.

After introduction of 4 kg of pChi750HL1 or pChi796HL1 obtained inParagraph 8 into 4×10⁶ YB2/0 (ATCC CRL1581) cells, the cells weresuspended in 40 ml of RPMI-1640-FCS(10) RPMI1640 medium (NissuiPharmaceutical) containing 10% of FCS, 1/40 volume of 7.5% NaHCO₃, 3% of200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycinsolution (Gibco; containing 5,000 units/ml penicillin and 5,000 μg/mlstreptomycin)!, and the suspension was distributed in 200-μl portionsinto wells of 96-well microtiter plates. After 24 hours of incubation at37° C. in a CO₂ incubator, G418 (Gibco) was added to a concentration of0.5 mg/ml and then incubation was continued for 1 to 2 weeks.Transformant colonies appeared, the culture fluid was recovered fromeach well in which the cells had grown to confluence and anenzyme-linked immunosorbent assay (ELISA) was conducted for anti-GM₂chimeric human antibody activity measurement.

Enzyme-linked immunosorbent assay (ELISA)

In a solution of 5 ng of phosphatidylcholine (Sigma) and 2.5 ng ofcholesterol (Sigma) in 2 ml of ethanol was dissolved 2 ng of GM₂(N-acetyl-GM₂ ; Boehringer Mannheim) or some other ganglioside. Thesolution or dilutions thereof were respectively distributed in 20-μlportions into wells of 96-well microtiter plates (Greiner) and, afterair drying, blocking was effected with PBS containing 1% BSA. Eachculture supernatant for each transformant, each purified mousemonoclonal antibody solution and each purified chimeric human antibodysolution were distributed in 50- to 100-μl portions into the wells andthe reaction was allowed to proceed at room temperature for 1 to 2hours. The wells were then washed with PBS, and 50 to 100 μl ofperoxidase-labeled antibody were added thereto followed by reaction atroom temperature for 1 to 2 hours. The wells were washed with PBS and anABTS substrate solution prepared by dissolving 550 mg of2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in0.1M citrate buffer (pH 4.2) and adding, just prior to use, hydrogenperoxide to a concentration of 1 μl/ml! was added in 50- to 100-μlportions to each well for color development, followed by OD₄₁₅measurement.

The clone showing the highest activity in ELISA among the clonesobtained gave a chimeric human anti-GM₂ antibody content of about 1.0μg/ml of culture fluid.

Cells of the clone showing the above-mentioned chimeric human anti-GM₂antibody activity were suspended in RPMI1640-FCS(10) medium containing0.5 mg/ml G418 and 50 nM methotrexate (hereinafter, "MTX") to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was performedat 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 50 nMMTX-resistant clones. At the time of confluence, the chimeric humananti-GM₂ antibody activity in each culture fluid was determined byELISA. The 50 nM MTX-resistant clone showing the highest activity amongthe clones obtained showed a chimeric human anti-GM₂ antibody content ofabout 5.0 μg/ml.

Cells of the above 50 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was carriedout at 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 200 nMMTX-resistant clones. At the time of confluence, each culture fluid wasassayed for chimeric human anti-GM₂ antibody activity by ELISA. The 200nM MTX-resistant clone showing the highest activity among the clonesobtained had a chimeric human anti-GM₂ antibody content of about 10μg/ml. The 200 nM MTX-resistant clones obtained from pChi750HL1 andpChi796HL1 were named transformants "KM966" (KM-796-derived chimerichuman antibody KM966-producing strain) and "KM967" (KM-750-derivedchimeric human antibody KM967-producing strain), respectively.

The following SDS-polyacrylamide gel electrophoresis (SDS-PAGE)confirmed that the above transformants KM966 and KM967 express therespective chimeric human anti-GM₂ antibodies.

The transformants KM966 and KM967 were each suspended in GIT medium(Nippon Pharmaceutical) containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml. Each suspension was distributed in100-ml portions into 175 cm² flasks (Greiner). Cultivation was performedat 37° C. in a CO₂ incubator for 5 to 7 days. At the time of confluence,the culture fluid was recovered. Treatment of about 1 liter of theculture fluid with Affi-Gel Protein A MAPS-II kit (Bio-Rad) gave about 5mg of a purified chimeric human anti-GM₂ antibody for each transformant.About 2 μg of the purified chimeric human anti-GM₂ antibody KM966 orKM967 was electrophoresed by the conventional method Laemmli: Nature,227, 680 (1970)! for molecular weight checking. The results are shown inFIG. 18. As shown in FIG. 18, both KM966 and KM967 gave an antibody Hchain molecular weight of about 50 kilodaltons and an antibody L chainmolecular weight of about 25 kilodaltons under reducing conditions,indicating the correctness in molecular weight of the H chain and Lchain expressed. For each of KM966 and KM967, the molecular weight ofthe chimeric human antibody under nonreducing conditions was about 150kilodaltons, indicating that the antibody expressed was composed of twoH chains and two L chains and was correct in size.

12. Expression of KM-603-derived chimeric human anti-GM₂ antibodies inSP2/0 cells

A 2-μg portion of the plasmid pChi603HMS1 or pChi603LMS1 obtained inParagraph 9 was introduced into 4×10⁶ cells of YB2/0 (ATCC CRL1581) byelectroporation in the same manner in Paragraph 11. The cells weresuspended in 40 ml of RPMI1640-FCS(10) and the suspension wasdistributed in 200-μl portions into wells of 96-well microtiter plates.After 24 hours of incubation in a CO₂ incubator at 37° C., G418 (Gibco)was added to a concentration of 0.5 mg/ml and incubation was continuedfor 1 to 2 weeks. Transformant colonies appeared. The culture fluid wasrecovered from confluent wells and the chimeric human anti-GM₂ antibodyactivity was measured by ELISA as described above. The clone showing thehighest chimeric human anti-GM₂ antibody activity among the clonesobtained gave a chimeric human anti-GM₂ antibody content of about 0.1μg/ml of culture fluid.

Cells of the clone showing the above-mentioned chimeric human anti-GM₂antibody activity were suspended in RPMI1640-FCS(10) medium containing0.5 mg/ml G418 and 50 nM MTX to a concentration of 1 to 2×10⁵ cells/mland the suspension was distributed in 2-ml portions into wells of24-well plates. Clones resistant to 50 nM MTX were induced by incubatingin a CO₂ incubator at 37° C. for 2 to 3 weeks. When confluence wasattained, the culture fluids were subjected to ELISA for chimeric humananti-GM₂ antibody activity measurement. Among the 50 nM MTX-resistantclones obtained, the clone showing the highest activity gave a chimerichuman anti-GM₂ antibody content of about 0.3 μg/ml of culture fluid.

Cells of the above 50 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspensions as distributedin 2-ml portions into well of 24-well plates. Clones resistant to 200 nMMTX were induced by following incubation in a CO₂ incubator at 37° C.for 2 to 3 weeks. When confluence was attained, the chimeric humananti-GM₂ antibody activity in the culture fluid was measured by ELISA.Among the 200 nM MTX-resistant clones obtained, the clone showing thehighest activity gave a chimeric human anti-GM₂ antibody content ofabout 0.5 μg/ml of culture fluid.

Cells of the above 200 nM MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 500 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspension was distributedin 2-ml portions into well of 24-well plates. Clones resistant to 500 nMMTX were induced following incubation in a CO₂ incubator at 37° C. for 1to 2 weeks. When confluence was attained, the chimeric human anti-GM₂antibody activity in the culture fluid was determined by ELISA. Amongthe 500 nM MTX-resistant clones obtained, the one showing the highestactivity gave a chimeric human anti-GM₂ antibody content of about 1.0μg/ml of culture fluid. This 500 nM MTX-resistant clone was named"transformant KM968".

The following SDS-PAGE confirmed the expression of a chimeric humananti-GM₂ antibody in the above transformant KM968.

Cells of the transformant KM968 were suspended in GIT medium (NipponPharmaceutical) containing 0.5 mg/ml G418 and 500 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml and the suspension was distributedin 100-ml portions into 175 cm² flasks (Greiner). Cultivation wasconducted in a CO₂ incubator at 37° C. for 5 to 7 days and, whenconfluence was attained, the culture fluid was recovered. Treatment ofabout 3.0 liters of the culture fluid with Affi-Gel Protein A MAPS-IIkit (Bio-Rad) gave about 1 mg of a purified chimeric human anti-GM₂antibody. About 2 μg of this purified chimeric human anti-GM₂ antibodyKM968 was electrophoresed by the conventional method Laemmli: Nature,227, 680 (1970)! for molecular weight checking. The results are shown inFIG. 19. Under reducing conditions, the molecular weight of the antibodyH chain was about 50 kilodaltons and the molecular weight of theantibody L chain was about 25 kilodaltons, thus confirming theexpression of the H chain and L chain having the correct molecularweight. Under nonreducing conditions, the molecular weight of thechimeric human antibody was about 150 kilodaltons, confirming that theantibody expressed was composed of two H chains and two L chains and wascorrect in size.

13. Reaction specificity of the chimeric human anti-GM₂ antibodies

The reactivities of the chimeric anti-GM₂ antibodies with gangliosideGM₁, N-acetyl-GM₂ (Boehringer Mannheim), N-glycolyl-GM₂, N-acetyl-GM₃,N-glycolyl-GM₃, GD_(1a), GD_(1b) (Iatron), GD₂, GD₃ (Iatron) and GQ_(1b)(Iatron) were examined by the technique of ELISA. The results are shownbelow in Table 1. GM₁ and GD_(1a) were purified from the bovine brain,N-glycolyl-GM₂ and N-glycolyl-GM₃ from the mouse liver, N-acetyl-GM₃from canine erythrocytes, and GD₂ from the cultured cell line IMR32(ATCC CCL127), by a known method J. Biol. Chem., 263, 10915 (1988)!.

As shown in Table 1, it was confirmed that the chimeric human anti-GM₂antibodies KM966 and KM967 specifically react with GM₂. The reactivityof KM966 was greater than that of KM967, however. On the contrary, KM968(KM-603-derived chimeric human antibody) did not show any reactivity forGM₂.

                  TABLE 1                                                         ______________________________________                                                   Binding activity of antibody (OD.sub.415)                          Ganglioside  KM966 (5 μg/ml)                                                                        KM967 (5 μg/ml)                                   ______________________________________                                        GM.sub.1     0.105       0.000                                                N-Acetyl-GM.sub.2                                                                          0.870       0.423                                                N-Glycolyl-GM.sub.2                                                                        0.774       0.065                                                N-Acetyl-GM.sub.3                                                                          0.002       0.000                                                N-Glycolyl-GM.sub.3                                                                        0.122       0.001                                                GD.sub.1a    0.004       0.000                                                GD.sub.1b    0.002       0.000                                                GD.sub.2     0.095       0.001                                                GD.sub.3     0.004       0.000                                                GQ.sub.1b    0.005       0.000                                                ______________________________________                                    

14. Reactivities of the chimeric human anti-GM₂ antibodies KM966 andKM967 with cancer cells (fluorescent antibody technique)

Suspended in PBS were 1×10⁶ cells of cultured human lung small cellcarcinoma cell line QC90 Cancer Res., 49, 2683 (1989)!, NCI-H69 (ATCCHTB119), NCI-H128 (ATCC HTB120), SBC-1 (JCRB 0816), SBC-2 (JCRB 0817),SBC-3 (JCRB 0818), SBC-5 (JCRB 0819), RERF-LC-MA (JCRB 0812), Lu-134-A-H(JCRB 0235), Lu-139 (RCB 469), Lu-130 (RCB 465), Lu-135 (RCB 468),Lu-134-B (RCB 467), Lu-140 (RCB 470), PC-6 Naito et al.: Gann to KagakuRyoho (Cancer and Chemotherapy), 5 (suppl.), 89 (1978)!, cultured humanlung squamous carcinoma cell line PC-1 Naito et al.: Gann to KagakuRyoho, 5 (suppl.), 89 (1978)!, PC-10 Naito et al.: Gann to Kagaku Ryoho,5 (suppl.), 89 (1978)!, Colo16 Moor et al.: Cancer Res., 35, 2684(1975)!, Calu-1 (ATCC HTB54), SK-LC-4 Proc. Natl. Acad. Sci. U.S.A., 85,4441 (1988)!, cultured human lung adenocarcinoma cell line PC-7 Hayataet al.: Hito Gansaibo no Baiyo (Human Cancer Cell Culture), 131 (1975)!,PC-9 Kinjo et al.: Brit. J. Cancer, 39, 15 (1979)!, PC-12 (ATCCCRL1721), RERF-LC-MS (JCRB 0081), HLC-1 (RCB 083), cultured human lunglarge cell carcinoma cell line PC-13 Ohya et al.: Tanpakushitsu,Kakusan, Koso (Protein, Nucleic Acid, Enzyme), 23, 697 (1978)!, Lu65(JCRB 0079), CALU-6 (ATCC HTB56), SK-LC-6 Proc. Natl. Acad. Sci. U.S.A.,85, 4441 (1988)!, cultured human neuroblastoma cell line YT-nu Ishikawaet al.: Acta Path. Jap., 27, 697 (1977)!, NAGAI Ishikawa et al.: ActaPath. Jap., 29, 289 (1979)!, NB-1 Ishikawa et al.: Acta Path. Jap., 27,697 (1977)!, IMR32 (ATCC CCL127), GOTO (JCRB 0612), NB-9 (RCB 477),SK-N-MC (ATCC HTB10), cultured human brain tumor (glioma) cell line P122EMBO J., 6, 2939 (1987)!, A172 (ATCC CRL1620), T98G (ATCC CRL1690),U-118MG (ATCC HTB15), cultured human leukemia cell line HSB-2 (ATCCCCL120.1), ATN-1, U-937 (ATCC CRL1593), HPB-ALL Ohya et al.:Tanpakushitsu, Kakusan, Koso, 23, 697 (1978)!, CCRF-SB (ATCC CCL120),KOPN-K Hanei et al.: Haigan (Lung Cancer), 25, 524 (1985)!, TYH Haranakaet al.: Int. J. Cancer, 36, 313 (1985)!, MOLT-3 (ATCC CRL1552), CCRF-CEM(ATCC CCL119), TALL-1 (JCRB 0086), NALL-1 Ohya et al.: Tanpakushitsu,Kakusan, Koso, 23, 697 (1978), CCRF-SB (JCRB 0032), THP-1 (ATCC TIB202),HEL92-1-7 (ATCC TIB180), cultured human maligant melanoma cell lineC24.32 (EP-A-0 493 686, KHm-3/P J. Natl. Cancer Inst., 59, 775 (1977)!or G361 (ATCC CRL1424). The suspension was placed in a microtube (Tref)and centrifuged (3,000 rpm, 2 minutes) to wash the cells, 50 μl of KM966or KM967 (50 μg/ml) was added, the mixture was stirred, and the reactionwas allowed to proceed at 4° C. for 1 hour. Then, the cells were washedthree times by centrifugation with PBS, 20 μl of fluoresceinisocyanate-labeled protein A (30-fold dilution; Boehringer MannheimYamanouchi) was added and, after stirring, the reaction was allowed toproceed at 4° C. for 1 hour. Then, the cells were washed three times bycentrifugation with PBS, then suspended in PBS and subjected to analysisusing flow cytometer EPICS Elite (Coulter). In a control run, the sameprocedure as described above was followed without adding the chimerichuman antibody. The results thus obtained are shown in Table 2. Thechimeric human antibody KM966 reacted with 9 (NCI-H128, SBC-1, SBC-3,SBC-5, Lu-139, Lu-130, Lu-135, Lu-134-B and Lu-140) of the 14 lung smallcell carcinoma cell lines, 2 (PC-10 and Calu-1) of the 5 lung squamouscarcinoma cell lines, 2 (PC-9 and RERF-LC-MS) of the 5 lungadenocarcinoma cell lines, 2 (PC-13 and SK-LC-6) of the 4 lung largecell carcinoma cell lines, 7 (YT-nu, NAGAI, NB-1, IMR32, GOTO, NB-9 andSK-N-MC) of the 7 neuroblastoma cell lines and 4 (P122, A172, T98G andU-118MG) of the 4 brain tumor (glioma) cell lines. On the other hand,the chimeric human antibody KM967 did not react with any of the culturedcell lines. The above results indicate that the chimeric human antibodyKM966 is useful in the diagnosis and treatment of brain tumors,peripheral nervous system tumors and lung cancer, among others.

                  TABLE 2                                                         ______________________________________                                                          KM966 (%) KM967 (%)                                         Cell line         (50 μg/ml)                                                                           (50 μg/ml)                                     ______________________________________                                        Lung small cell carcinoma                                                                       9/14   (64)   0/14   (0)                                    Lung squamous cell                                                                              2/5    (40)   0/5    (0)                                    carcinoma                                                                     Lung adenocarcinoma                                                                             2/5    (40)   0/5    (0)                                    Lung large cell carcinoma                                                                       2/4    (50)   0/4    (0)                                    Neuroblastoma     7/7    (100)  0/7    (0)                                    Brain tumor (glioma)                                                                            4/4    (100)  0/4    (0)                                    Leukemia          0/14   (0)    0/14   (0)                                    Malignant melanoma                                                                              0/3    (0)    0/3    (0)                                    ______________________________________                                    

15. In vitro antitumor activity of the chimeric human anti-GM₂ antibodyKM966: complement dependent cytotoxicity (CDC)

(1) Preparation of target cells

The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO orA172, cultured in RPMI1640 medium supplemented with 10% FCS, wereadjusted to a cell concentration of 5×10⁶ cells/ml, Na₂ ⁵¹ CrO₄ wasadded to a concentration of 100 μCi/5×10⁶ cells, then the reaction wasallowed to proceed at 37° C. for 1 hours, and the cells were washedthree times with the medium. The cells were then allowed to stand in themedium at 4° C. for 30 minutes for spontaneous dissociation and then,after centrifugation, the medium was added to adjust the cellconcentration to 1×10⁶ cells/ml.

(2) Preparation of the complement

Sera from three healthy subjects were combined and used as a complementsource.

(3) CDC activity measurement

The chimeric human anti-GM₂ antibody KM966 or mouse anti-GM₂ antibodyKM696 (FERM BP-3337) was added to wells of 96-well U-bottom plateswithin the final concentration range of 0.5 to 50 μg/ml and then 5×10⁴cells/well of the target cells prepared in (1) were added. The reactionwas allowed to proceed at room temperature for 30 minutes. Aftercentrifugation, the supernatants were discarded, 150 μl of the humanserum obtained in (2) was added to each well (final concentration 15%v/v), and the reaction was allowed to proceed at 37° C. for 1 hour.After centrifugation, the amount of ⁵¹ Cr in each supernatant wasdetermined using a gamma counter. The amount of spontaneouslydissociated 51Cr was determined by adding to the target cells the mediumalone in lieu of the antibody and complement solutions and measuring theamount of ⁵¹ Cr in the supernatant in the same manner as mentionedabove. The total amount of dissociated ⁵¹ Cr was determined by adding tothe target cells 5N sodium hydroxide in lieu of the antibody andcomplement solutions and measuring the amount of ⁵¹ Cr in thesupernatant in the same manner as mentioned above. The CDC activity wascalculated as follows: ##EQU1##

The results thus obtained are shown in FIGS. 20 to 23. It was shown thatthe chimeric human antibody KM966 show CDC activity against all thecells tested.

16. In vitro antitumor activity of the chimeric human anti-GM₂ antibodyKM966: antibody dependent cell mediated cytotoxicity (ADCC)

(1) Preparation of target cells

The target cells SBC-3, Lu-135, PC-10, RERF-LC-MS, PC-13, NAGAI, GOTO orA172, cultured in RPMI1640 medium supplemented with 10% FCS, wereadjusted to a cell concentration of 1×10⁶ cells/ml, Na₂ ⁵¹ CrO₄ wasadded to a concentration of 50 μCi/1×10⁶ cells, then the reaction wasallowed to proceed at 37° C. for 1 hour, and the cells were washed threetimes with the medium. The cells were then allowed to stand in themedium at 4° C. for 30 minutes for spontaneous dissociation and then,after centrifugation, the medium was added to adjust the cellconcentration to 2×10⁵ cells/ml.

(2) Preparation of effector cells

Human venous blood (25 ml) was collected, 0.5 ml of heparin sodium(Takeda Chemical Industries; 1,000 units/ml) was added, and the mixturewas gently stirred. This mixture was centrifuged (1,500 to 1,800 g, 30minutes) using Polymorphprep (Nycomed Pharma AS), the lymphocyte layerwas separated and washed three times by centrifugation with RPMI-1640medium (15,00 to 1,800 g, 15 minutes), and the cells were suspended inRPMI1640 medium supplemented with 10% FCS (5×10⁶ cells/ml) for use aseffector cells.

(3) ADCC activity measurement

The chimeric human anti-GM₂ antibody KM966 or mouse anti-GM₂ antibodyKM696 were added to wells of 96-well U-bottom plates within the finalconcentration range of 0.05 to 5 μg/ml and then 50 μl (1×10⁴ cells/well)of the target cell suspension prepared in (1) and 100 μl (5×10⁵cells/well) of the effector cell suspension prepared in (2) were addedto each well (the ratio between the effector cells and target cellsbeing 50:1). The reaction was allowed to proceed at 37° C. for 4 hoursand, after centrifugation, the amount of ⁵¹ Cr in each supernatant wasmeasured using a gamma counter. The amount of spontaneously dissociated⁵¹ Cr was determined by adding to the target cells the medium alone inlieu of the antibody and effector cells and measuring the amount of ⁵¹Cr in the supernatant in the same manner as mentioned above. The totalamount of dissociated ⁵¹ Cr was determined by adding to the target cells5N sodium hydroxide in lieu of the antibody and effector cells andmeasuring the amount of ⁵¹ Cr in the supernatant in the same manner asmentioned above. The ADCC activity was calculated as follows: ##EQU2##

The results thus obtained are shown in FIGS. 24 to 27. The chimericantibody KM966 showed ADCC activity against all the cells whereas themouse anti-GM₂ antibody KM696 showed no or low ADCC activity. The aboveresults indicate that the chimeric human antibody KM966 is moreeffective in the treatment of human cancer than the mouse antibodyKM-696.

REFERENCE EXAMPLE 1 Construction of the vector pChiIgLA1 for chimerichuman antibody L chain expression

1. Isolation of the KM50 cell-derived immunoglobulin H chain promoterand enhancer genes

(1) Preparation of chromosomal DNAs from KM50 cells, P3U1 cells and ratkidney

Chromosal DNAs were prepared by the conventional method Maniatis et al.(ed.): Molecular Cloning, 1989, p. 9.16!, as follows.

KM50 cells (1.2×10⁸ cells), P3U1 cells (ATCC CRL1597) (2×10⁸ cells) anda rat kidney sample (frozen at -80° C. and then smashed to a sufficientextent using a wooden hammer) (1.6 g) were suspended in 2 ml of 10 mMTris-hydrochloride buffer (pH 7.5) containing 150 mM sodium chloride and10 mM ethylenediaminetetraacetic acid disodium salt (hereinafter,"EDTA"), 0.8 mg of proteinase K (Sigma) and 10 mg of sodium laurylsulfate (hereinafter, "SDS"), were added to each suspension, and thesuspension was incubated at 37° C. for 10 hours. Then, each mixture wasextracted once with an equal volume of phenol, twice with an equalvolume of chloroform and then once with an equal volume of ether, anddialyzed for 10 hours against 10 mM Tris-hydrochloride buffer (pH 7.5)containing 1 mM EDTA. The DNA solution was recovered from the dialysistube and ribonuclease A (Sigma) was added to the solution to a finalconcentration of 20 μg/ml. Each resultant solution was incubated at 37°C. for 6 hours for sufficient decomposition of RNA, 15 mg of SDS and 1mg of proteinase K were then added and the mixture was incubated at 37°C. for 10 hours. The mixture was then extracted twice with an equalvolume of phenol, twice with an equal volume of chloroform and twicewith an equal volume of ether and then dialyzed for 10 hours against 10mM Tris-hydrochloride buffer (pH 7.5) containing 1 mM EDTA. The DNAsolution was recovered from the dialysis tube for use as a chromosomalDNA sample. DNA concentration measurement in terms of the absorbance at260 nm revealed that the yield of chromosomal DNA from 1.2×10⁸ KM50cells was 1.6 mg, that from 2×10⁸ P3U1 cells 1.5 mg, and that from 1.6 gof rat liver 1.9 mg.

(2) Identification of the active-form immunoglobulin H chain gene inKM50 cells by Southern blotting

The KM50 cell, p3U1 cell and rat kidney chromosomal DNAs obtained in (1)(3 μg each) were dissolved in 25 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride,15 units of XbaI (Takara Shuzo; hereinafter the restriction enzymes usedwere products of Takara Shuzo) was added and incubation was carried outat 37° C. for 2 hours for effecting cleavage at the XbaI sites. Eachreaction mixture was subjected to agarose gel electrophoresis, then DNAtransfer onto a nitrocellulose filter was effected by the method ofSouthern et al. J. Mol. Biol., 98, 503 (1975)! and hybridization wascarried out by the conventional method Kameyama et al.: FEBS Letters,244, 301-306 (1989)! using the mouse JH probe described in thelast-cited reference. The KM50 cell DNA alone gave a band at a sitecorresponding to about 9.3 kb. Therefore, the immunoglobulin XbaIfragment DNA was considered to code for the active-form immunoglobulin Hchain gene in KM50 cells.

(3) Construction of a KM50 cell genomic DNA library

A 60-μg portion of the KM50 cell-derived chromosomal DNA obtained in (1)was dissolved in 250 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 150 unitsof XbaI was added, and incubation was conducted at 37° C. for 2 hoursfor causing cleavage at the XbaI sites. The reaction mixture wasfractionated by agarose gel electrophoresis and a KM50 cell-derived 9.3kb DNA fraction sample (about 2 μg) was recovered using, for example,the DEAE paper method Maniatis et al. (ed.): Molecular Cloning, 1989, p.6.24!. Separately, 3 μg of Lambda ZAP (Stratagene), for use as thevector, was dissolved in 200 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 50units of XbaI was added, and the mixture was incubated at 37° C. for 2hours to effect cleavage at the XbaI sites. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, whereby about 3 kg of DNA was recovered. This DNA wasdissolved in 100 μl of 100 mM Tris-hydrochloride buffer (pH 7.5), 1 unitof alkaline phosphatase (Takara Shuzo) was added, dephosphorylation waseffected at the restriction enzyme cleavage ends of the vector DNA. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation, whereby 2 μg of DNA was recovered. This DNAwas dissolved in 10 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 1 mM EDTA for use as a vector sample. Two tenths μg of thevector DNA sample and 0.2 μg of the KM50 cell-derived 9.3 kb DNA samplewere dissolved in 5 μl of T4 ligase buffer, 175 units of T4 ligase(Takara Shuzo) was added, and the mixture was incubated at 4° C. for 3days. A 2-μl portion of this mixture was packaged into the lambda phageby the conventional method Maniatis et al. (ed.): Molecular Cloning,1989, p. 2.95! using Giga Pak Gold (Stratagene), and the packagingmixture was used to transfect Escherichia coli BB4 to give 200,000 phageclones. Among them, 100,000 clones were fixed on a nitrocellulose filterby the conventional method Maniatis et al. (ed.): Molecular Cloning,1989, p. 2.112!.

(4) Selection of a recombinant DNA containing the gene for the H chainvariable region of an immunoglobulin occurring as an active form in KM50cells (anti-human serum albumin)

From among the phage library composed of 100,000 clones, as constructedin (3), two clones firmly associable at 65° C. with the ³² P-labeledmouse JH probe labeled by the method of Kameyama et al. FEBS Letters,44, 301-306 (1989)!! were isolated. The phage DNA was recovered fromthem by the conventional method Maniatis et al. (ed.): MolecularCloning, 1989, p. 2.118-2.169!, whereupon the 9.3 kb XbaI fragment ofthe KM50 cell-derived chromosomal DNA was found to have been insertedtherein.

(5) Base sequence of the gene for the H chain variable region of theimmunoglobulin occurring as an active form in KM50 cells (anti-humanserum albumin)

For the two clones obtained in (4), restriction enzyme cleavage mapswere prepared by conducting digestion using various restriction enzymes,whereby it was revealed that the same DNA fragment (9.3 kb) had beeninserted therein (FIG. 28). Therefore, those portions of this 9.3 kb DNAfragment which were supposed to be coding for the rat immunoglobulin Hchain promoter region and variable region were sequenced by the methodof Sanger Sanger et al.: Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977);AMERSHAM M13 cloning and sequencing handbook!. In SEQ ID NO:16, theportion containing the octamer sequence such as ATGCAAAT and the TATAbox sequence such as TTGAAAA is considered to be the immunoglobulinpromoter region.

2. Construction of heterologous protein expression vectors using thepromoter and enhancer for the H chain variable region gene for animmunoglobulin occurring as an active form in KM50 cells (anti-humanserum albumin)

(1) Construction of pKMB11

A 1-μg portion of the 9.3 kb immunoglobulin H chain variable region genefragment obtained in Paragraph 1 (5) was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units each of BglII and HindIII wereadded, and the mixture was incubated at 37° C. for 2 hours for causingcleavage at the BglII and HindIII sites. The reaction mixture wassubjected to agarose gel electrophoresis and 0.01 μg of a DNA fragmentcontaining the 0.8 kb immunoglobulin promoter was recovered. Then, 1 μgof the plasmid pBR322-BglII Kuwana et al.: FEBS Letters, 219, 360(1987)! was dissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of BglII and 10 units of HindIII were added, and the mixture wasincubated at 37° C. for 2 hours to effect cleavage at the BglII andHindIII sites. The reaction mixture was subjected to agarose gelelectrophoresis and a DNA fragment of about 4.2 kb in size wasrecovered. The thus-obtained pBR322-BglII-derived DNA fragment (about4.2 kb, 0.1 μg) and immunoglobulin promoter-containing DNA fragment(0.01 μg) were dissolved in 20 Mil of T4 ligase buffer, 175 units of T4DNA ligase (Takara Shuzo) was added, and the mixture was incubated at 4°C. for 1 day. The reaction mixture was used to transform Escherichiacoli HB101 (J. Mol. Biol., 41, 459 (1969)! by the method of Scott et al.Masaru Shigesada: Saibo Kokagu (Cell Engineering), 2, 616 (1983)! togive an Ap-resistant colony. The recombinant plasmid DNA was recoveredfrom this colony. Plasmid pKMB11, shown in FIG. 29, was thus obtained.

(2) Construction of pKMD6

For providing an appropriate restriction enzyme site downstream from theimmunoglobulin promoter, the plasmid pKMB11 constructed in (1) wasdigested at the NcoI site using the nuclease BAL31. Thus, 10 μg of theplasmid pKMB11 was dissolved in 100 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 6 mM magnesium chloride and 50 mM potassiumchloride, 30 units of NcoI was added, and the mixture was incubated at37° C. for 2 hours to effect cleavage at the NcoI site. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the whole amount of the DNA fragment wasdissolved in 100 μl of BAL31 buffer 20 mM Tris-hydrochloride buffer (pH8.0) containing 600 mM sodium chloride, 12 mM calcium chloride, 12 mMmagnesium chloride and 1 mM EDTA!, 0.25 unit of BAL31 Bethesda ResearchLaboratories (BRL)!! was added, and digestion was carried out at 37° C.for 5 seconds. The reaction was terminated by extraction with phenol andsubjected to chloroform extraction and then to ethanol precipitation,and 1 μg of DNA was recovered. A 0.1-μg portion of this DNA and 0.01 μgof a synthetic DNA linker (SalI) were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101 by the method of Scott et al. An Ap-resistantcolony was obtained and the recombinant plasmid DNA was recovered fromthis colony to give the plasmid pKMD6 shown in FIG. 30. For thisplasmid, the portion of BAL31 digestion was sequenced by the method ofSanger, whereupon deletion was found to the third base (303rd base inSEQ ID NO:16) toward the upstream of the initiation codon ATG forimmunoglobulin.

(3) Construction of pEPKMA1, pEPKMB1 and pAGE501

The original immunoglobulin promoter and enhancer are positionallyseparated. Therefore, it was necessary to construct a vector containingthe promoter and enhancer connected to each other for use of said vectoras a heterologous protein expression vector. Accordingly, the followingprocedure was followed.

Thus, 1 μg of the 9.3 kb immunoglobulin H chain variable region geneobtained in Paragraph 1 (5) was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of EcoRV and 10 units of XbaI wereadded, and the mixture was incubated at 37° C. for 2 hours for causingcleavage at the EcoRV and XbaI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.1 μg of a DNA fragment (about 1 kb)containing the immunoglobulin enhancer region was recovered. Separately,1 μg of the plasmid pKMD6 obtained in (2) was dissolved in 100 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of BglII was added, and the mixturewas incubated at 37° C. for 2 hours to effect cleavage at the BglIIsite. After phenol-chloroform extraction, the DNA was precipitated withethanol and dissolved in a total of 40 μl of DNA polymerase I buffer, 6units of Escherichia coli-derived DNA polymerase I Klenow fragment wasadded, and the reaction was allowed to proceed at 16° C. for 90 minutesfor rendering the 5' protruding ends formed upon BglII digestionblunt-ended. The reaction was terminated by extraction with phenol, themixture was extracted with chloroform and then subjected to ethanolprecipitation, the DNA obtained was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was added, and themixture was incubated at 37° C. for 2 hours to effect cleavage at theHindIII site. The reaction mixture was subjected to agarose gelelectrophoresis and 0.1 μg of a DNA fragment (about 0.8 kb) containingthe immunoglobulin promoter region was recovered. Then, 0.2 μg of theplasmid pUC18 Messing: Methods in enzymology 101, 20 (1983)! wasdissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 unitsof HindIII and 10 units of XbaI were added, and the mixture wasincubated at 37° C. for 2 hours for causing cleavage at the HindIII andXbaI sites. The reaction mixture was subjected agarose gelelectrophoresis and 0.1 μg of a DNA fragment of about 2.7 kb in size wasrecovered. The thus-obtained pPKMD6-derived 0.8 kb DNA fragment (0.1μg), immunoglobulin enhancer region-containing DNA fragment (0.02 μg)and pUC18 (0.1 μg) were dissolved in 20 μl of T4 ligase buffer, 175units of T4 DNA ligase was added, and the mixture was incubated at 4° C.for 1 day. The reaction mixture was used to transform Escherichia coliHB101 to give an Ap-resistant colony. The recombinant plasmid DNA wasrecovered from this colony to give pEPKMA1 shown in FIG. 31.

Then, 1 μg of the plasmid pEPKMA1 was dissolved in 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of XbaI was added, and the mixturewas incubated at 37° C. for 2 hours for causing cleavage at the XbaIsite. After phenol-chloroform extraction, the resultant DNA fragment wasprecipitated with ethanol and dissolved in a total of 40 μl of DNApolymerase I buffer, 6 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was allowed to proceed at16° C. for 90 minutes for rendering the cohesive ends formed upon XbaIdigestion blunt-ended. The reaction was terminated by extraction withphenol and, after chloroform extraction, the DNA fragment was recoveredby ethanol precipitation. This DNA fragment and a synthetic DNA linkerXhoI (Takara Shuzo) (0.01 μg) were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101 to give an Ap-resistant colony. The recombinantplasmid DNA was recovered from this colony to give pEPKMB1 shown in FIG.32.

Then, the SV40 early gene promoter and enhancer regions (hereinafterabbreviated as PSE) of the heterologous gene expression vector pAGE107for use in animals Miyaji et al.: Cytotechnology, 3, 133-140 (1990)!were replaced with the KM50-derived immunoglobulin H chain promoter andenhancer (hereinafter abbreviated as PIE) of PEPKMB1 in the followingmanner.

One μg of the plasmid pAGE107 was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 150 mM sodium chloride, 10 units of SalI and 10 units of XhoI wereadded, and the mixture was incubated at 37° C. for 2 hours to-effectcleavage at the SalI and XhoI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.5 μg of a DNA fragment (about 5.95kb) containing the G418 resistance gene, among others, was recovered.Then, 1 μg of the plasmid PEPKMB1 was dissolved in 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 150 mM sodium chloride, 10 units of SalI and 10 units of XhoI wereadded, and the mixture was incubated at 37° C. for 2 hours to effectcleavage at the SalI and XhoI sites. The reaction mixture was subjectedto agarose gel electrophoresis and 0.1 μg of a DNA fragment (about 1.7kb) containing the immunoglobulin promoter and enhancer regions wasrecovered. The thus-obtained obtained pAGE107-derived 5.95 kb DNAfragment (0.1 μg) and immunoglobulin promoter and enhancerregion-containing DNA fragment (0.02 μg) were dissolved in 20 μl of T4ligase buffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli HB101. An Ap-resistant colony was isolated and therecombinant plasmid DNA was recovered therefrom to give pAGE501 shown inFIG. 33.

(4) Construction of pAGE109

A plasmid, pAGE109, derived from pAGE106 by deletion of one of the twoEcoRI sites in pAGE106 was constructed as follows.

Thus, 2 μg of the heterologous gene expression vector pAGE106 for use inanimal cells es described in EP-A-0 405 285 was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride; 10 units each of EcoRI and SacI were furtheradded, and digestion was conducted at 37° C. for 4 hours. The reactionmixture was fractionated by agarose gel electrophoresis and about 1.5 μgof a DNA fragment (4.3 kb) resulting from cleavage of pAGE106 with EcoRIand SacI and containing the SV40 early gene promoter and G418 resistancegene was recovered. Then, this DNA fragment was dissolved in a total of40 μl of DNA polymerase I buffer, 5 units of Escherichia coli-derivedDNA polymerase I large fragment was added, and the reaction wasconducted at 16° C. for 2 hours for rendering the 3' protruding endsformed upon SalI digestion and the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was dissolved in 20 μl of T4 ligase buffer; 350 units of T4DNA ligase was further added to the mixed solution, and ligation wascarried out at 4° C. for 4 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to give the plasmidpAGE109 shown in FIG. 34.

(5) Construction of pAGE502

For replacing the SV40 promoter and enhancer of pAGE107 with theimmunoglobulin H chain promoter and enhancer, a plasmid named pAGE502was constructed as follows.

Two μg of pAGE107 described in EP-A-0 405 285 was aided to 100 μl of 10mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon HindIIIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of XhoI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 5.95 kb),resulting from cleavage of pAGE107 with XhoI and HindIII and containingthe G418 resistance gene and Ap resistance, was recovered.

Two μg of pAGE501 obtained in (3) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 175 mM sodium chloride, 10 units of SalI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon SalIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of XhoI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 0.2 μg of a DNA fragment (1.8 kb) resultingfrom cleavage of pAGE501 with XhoI and SalI and containing the KM50 cellimmunoglobulin H chain promoter and enhancer was recovered.

Then, 0.1 μg of the HindIII-XhoI fragment (about 5.95 kb) of pAGE107 asobtained above and 0.1 μg of the SalI-XhoI fragment (about 1.8 kb) ofpAGE501 were dissolved in a total of 20 μl of T4 ligase buffer; 350units of T4 DNA ligase was added to the solution, and the mixture wasincubated at 4° C. for 1 day. The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101 to give the plasmid pAGE502shown in FIG. 35.

(6) Construction of pAGE503

A plasmid named pAGE503 derived from pAGE502 by deletion of one of thetwo EcoRI sites was constructed as follows.

Two μg of pAGE109 obtained in (4) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride; 10 units of HindIII and 10 units of ClaI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.2 μg of a DNA fragment (about 1 kb) resulting from cleavage ofpAGE109 with ClaI and HindIII and containing the poly-A signal gene forthe beta globulin and SV40 early genes was recovered.

Then, 2 μg of pAGE502 obtained in (5) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII and 10 units of ClaI werefurther added, and digestion was conducted at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 1 μg of a DNA fragment (about 6.1 kb) resulting from cleavage ofpAGE502 with HindIII and ClaI and containing the KM50 cellimmunoglobulin H chain promoter and enhancer genes, the Ap resistancegene and the G418 resistance gene was recovered by the DEAE papermethod. Then, 0.1 μg of the HindIII-ClaI fragment (about 1 kb) ofpAGE109 as obtained above and 0.1 μg of the HindIII-ClaI fragment (about6.1 kb) of pAGE502 as obtained above were dissolved in a total of 20 μlof T4 ligase buffer, 350 units of T4 DNA ligase was added to thesolution, and the mixture was incubated at 4° C. for 1 day. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pAGE503 shown in FIG. 36 was obtained.

(7) Construction of pSE1d1

A plasmid named pSE1d1 was constructed by introducing the dhfr gene intopAGE107, as follows.

Two μg of pAGE107 described in EP-A-0 405 285 was added to 100 μl of 100mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of EcoRI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10units of HindIII was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 5.6 kb)resulting from cleavage of pAGE107 with EcoRI and HindIII and containingthe G418 resistance gene and Ap resistance gene was recovered.

Two μg of pSV2-dhfr Subramani et al.: Mol. Cell. Biol., 1, 854 (1981)!was added to 100 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 unitsof BglII was further added, and digestion was carried out at 37° C. for4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, the precipitate wasdissolved in a total of 40 μl of DNA polymerase I buffer, 5 units ofEscherichia coli-derived DNA polymerase I Klenow fragment was added, andthe reaction was conducted at 16° C. for 2 hours for rendering the 5'protruding ends formed upon BglII digestion blunt-ended. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the precipitate was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of apSV2-dhfr DNA fragment (0.76 kb) resulting from cleavage with BglII andHindIII and containing the dehydrofolate reductase (dhfr) gene wasrecovered.

Then, 0.1 μg of the HindIII=EcoRI fragment (about 5.6 kb) of pAGE107, asobtained above, and 0.1 μg of the BglII-HindIII fragment (about 0.76 kb)of pSV2-dhfr, as obtained above, were dissolved in a total of 20 μl ofT4 ligase buffer; 350 units of T4 DNA ligase was added to the solution,and the mixture was incubated at 4° C. for 1 day. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe plasmid pSE1d1 shown in FIG. 37 was obtained.

(8) Construction of pSE1d2

A plasmid named pSE1d2 was constructed by deleting the HindIII cleavagesite from pSE1d1, as follows.

Thus, 2 μg of pSE1d1 obtained in (7) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of HindIII was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon HindIIIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was dissolved in 20 μl of T4 ligase buffer, 350 units of T4DNA ligase was added to the solution, and the mixture was incubated at4° C. for 1 day. The thus-obtained recombinant plasmid DNA was used totransform Escherichia coli HB101 and the plasmid pSE1d2 shown in FIG. 38was obtained.

(9) Construction of pIg1SE1d2

A plasmid named pIg1SE1d2 was constructed by introducing the dhfr geneinto pAGE503, as follows.

Two μg of pAGE503 obtained in (6) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units of ClaI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon ClaIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 50 mM sodium chloride; 10units of MluI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1 μg of a DNA fragment (about 5.4 kb)resulting from cleavage of pAGE503 with ClaI and MluI and containing theKM50 immunoglobulin H chain promoter and enhancer was recovered.

Then, 2 μg of pSE1d2 obtained in (8) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of XhoI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was conducted at 16° C.for 2 hours for rendering the 5' protruding ends formed upon XhoIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, theprecipitate was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH7.5) containing 6 mM magnesium chloride and 100 mM sodium chloride, 10units of MluI was further added, and digestion was effected at 37° C.for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1 μg of a DNA fragment (about 3.8 kb)resulting from cleavage of pSE1d2 with XhoI and MluI and containing thedhfr gene was recovered.

Then, 1 μg of the ClaI-MluI fragment (about 5.4 kb) of pAGE503 asobtained above and 1 μg of the XhoI-MluI fragment (about 3.8 kb) ofpSE1d2 as obtained above were dissolved in a total of 20 μl of T4 ligasebuffer, 350 units of T4 DNA ligase was added to the solution, and themixture was incubated at 4° C. for 1 day. The thus-obtained recombinantplasmid DNA was used to transform Escherichia coli HB101 and the plasmidpIg1SE1d2 shown in FIG. 39 was obtained.

(10) Construction of pIg1SE1d3

A plasmid named pIg1SE1d3 was constructed by deleting the ApaI cleavagesite from pIg1SE1d2, as follows.

Two μg of pIg1SE1d2 obtained in (9) was added to 100 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chloride,10 units of ApaI was further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, the precipitate wasdissolved in a total of 40 μl of DNA polymerase I buffer, 5 units ofEscherichia coli-derived DNA polymerase I Klenow fragment was added, andthe reaction was carried out at 16° C. for 2 hours for rendering the 3'protruding ends formed upon ApaI digestion blunt-ended. The reactionmixture was subjected to phenol-chloroform extraction and then toethanol precipitation, the precipitate was dissolved in 20 μl of T4ligase buffer, 350 units of T4 ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe plasmid pIg1SE1d3 shown in FIG. 40 was obtained.

(11) Construction of pIg1SE1d4

For providing pIg1SE1d3 with a cloning site between the HindIII cleavagesite and EcoRI cleavage site, a plasmid named pIg1SE1d4 was constructedcontaining the synthetic DNA defined by SEQ ID NO:17 as an insert, asfollows.

Two μg of pIg1SE1d3 obtained in (10) was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 50 mM sodium chloride, 10 units each of HindIII and EcoRI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 1 μg of a DNA fragment (about 9.2 kb) resulting from cleavage ofpIg1SE1d3 with HindIII and EcoRI and containing the KM50 cellimmunoglobulin H chain promoter, enhancer, Ap resistance gene, G418resistance gene and dhfr gene was recovered.

Then, 0.1 μg of the HindIII-EcoRI fragment (about 9.2 kb) of pIg1SE1d3as obtained above and 10 ng of the synthetic DNA (SEQ ID NO:17) were atotal of 20 μl of T4 ligase buffer, 350 units of T4 DNA ligase was addedto the solution, and the mixture was incubated at 4° C. for 1 day. Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 and the plasmid pIg1SE1d4 shown in FIG. 41 was obtained.

3. Preparation of the Moloney mouse leukemia virus long terminal repeat(hereinafter abbreviated as "MoLTR")

It is known that MoLTR has promoter and enhancer activity Kuwana et al.:Biochem. Biophys. Res. Commun., 149, 960 (1987)!. Therefore, for usingMoLTR as a promoter and enhancer in vectors for chimeric human antibodyexpression, a plasmid, pPMOL3, containing MoLTR was constructed asfollows.

Three μg of pPMOL1 described in JP-A-1-63394 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chlorideand 6 mM 2-mercaptoethanol, 10 units of ClaI was further added, anddigestion was carried out at 37° C. for 4 hours. The reaction mixturewas subjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived DNA polymeraseI Klenow fragment was added, and the reaction was carried out at 16° C.for 2 hours for rendering the 5' protruding ends formed upon ClaIdigestion blunt-ended. The reaction was terminated by extraction withphenol, the reaction mixture was subjected to chloroform extraction andthen to ethanol precipitation, and 2 μg of a DNA fragment was recovered.This DNA fragment and 0.01 μg of a synthetic DNA linker XhoI (TakaraShuzo) were dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNAligase was added, and the mixture was incubated at 4° C. for 1 day. Thereaction mixture was used to transform Escherichia coli HB101 and theplasmid pPMOL2 shown in FIG. 42 was obtained. Then, 3 μg of pPMOL2 wasadded to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7mM magnesium chloride, 10 mM sodium chloride and 6 mM 2-mercaptoethanol,10 units of SmaI was further added, and digestion was conducted at 37°C. for 4 hours. The reaction mixture was subjected to phenol-chloroformextraction and then to ethanol precipitation, and 2 μg of a DNA fragmentwas recovered. This DNA fragment and 0.01 μg of a synthetic DNA linker(EcoRI; Takara Shuzo) were dissolved in 20 μl of T4 ligase buffer, 175units of T4 DNA ligase was added, and the mixture was incubated at 4° C.for 1 day. The reaction mixture was used to transform Escherichia coliHB101 and the plasmid pPMOL3 shown in FIG. 43 was obtained.

4. Cloning of the human immunoglobulin IgGl H chain constant region(Cγ1) cDNA and L chain constant region (Cκ) cDNA

(1) Isolation of mRNA from the chimeric antibody producer cell lineSP2-PC Chimera-1

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitrogen, mRNA (6.2 μg) was isolated from 1×10⁸ cellsof the chimeric antibody producer cell line SP2-PC Chimera-1 describedin FEBS Letters, 244, 301-306 (1989) and capable of producing a chimericantibody having anti-phosphorylcholine activity.

(2) Construction of an SP2-PC Chimera-1 cDNA library and cloning of thehuman immunoglobulin H chain constant region (Cγ1) cDNA and L chainconstant region (Cκ) cDNA

Starting with 2 μg of the mRNA obtained in (1) and using cDNA SynthesisKit (product number 27-9260-01) manufactured by Pharmacia, EcoRI adapterjoining was performed, followed by phosphorylation. The cDNA solutionobtained was subjected to phenol-chloroform extraction and then toethanol precipitation, and 4 μg of cDNA was recovered. This cDNA wasdissolved in 20 μl of sterilized water and then fractionated by agarosegel electrophoresis, and about 0.3 μg each of two DNA fragments, about1.8 kb and about 1.0 kb in size, were recovered.

Then, 5 μg of the vector pUC18 was added to 100 μl of 100 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 50 units of EcoRI was further added, anddigestion was carried out at 37° C. for 4 hours for cleaving the pUC18DNA at the EcoRI site. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation, andabout 3 μg of a DNA fragment resulting from cleavage of pUC18 at theEcoRI site thereof was recovered.

Then, 0.1 μg of the EcoRI fragment (about 2.7 kb) of pUC18 as obtainedabove and 0.1 μg each of the 1.8 kb and 1.0 kb cDNA fragments preparedfrom SP2-PC Chimera-1 cells were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was effected at 4° C. for 24 hours.

The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli LE392. About 3,000 colonies obtained were fixed onto anitrocellulose filter. From among the strains firmly bound at 65° C. toprobes prepared by labeling the human immunoglobulin constant regionchromosomal genes (IgGl H chain constant region Cγ1 and L chain constantregion Cκ) Kameyama et al.: FEBS Letters, 244, 301 (1989)! with ³² p, aplasmid (pPCVHhCGI1) associable with Cγ1 and another (pPCVLhCK1)associable with Cκ were isolated.

(3) Introduction of an EcoRV site into the human Igκ chain constantregion gene

An EcoRV site was introduced into the human Igκ chain constant region ata site near the 5' end thereof by site-directed mutagenesis using a kit(catalog number Q6210) manufactured by Promega. The plasmid pPCVLhCK1 (2μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 50 mM sodium chloride, 10 unitsof EcoRI and 10 units of KpnI were further added, and digestion wasconducted at 37° C. for 4 hours. The reaction mixture was fractionatedby agarose gel electrophoresis and about 0.2 μg of a DNA fragment (about0.8 kb) resulting from cleavage of pPCVLhCK1 with KpnI and EcoRI andcontaining the human immunoglobulin L chain constant region gene wasrecovered.

Then, 2 μg of pSELECT1 (a kit manufactured by Promega; catalog numberQ6210) was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 50 mM sodium chloride, 10 unitseach of EcoRI and KpnI were further added, and digestion was carried outat 37° C. for 4 hours. The reaction mixture was fractionated by agarosegel electrophoresis and about 1 μg of a DNA fragment (about 5.7 kb)resulting from cleavage of pSELECT1 with EcoRI and KpnI was recovered.

Then, 0.1 μg of the EcoRI-KpnI fragment (about 0.8 kb) of pPCVLhCK1 asobtained above and 0.1 μg of the EcoRI-KpnI fragment (about 5.7 kb) ofpSELECT1 as obtained above were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was effected at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli JM109 andthe plasmid pchCKA7 shown in FIG. 44 was obtained.

Then, using pchCKA7 (SEQ ID NO:49) and using the synthetic DNA definedby SEQ ID NO:18 as a mutagenic primer, the sequence covering the 12thbase to 14 base from the N terminus of the human immunoglobulin L chainconstant region, namely ACC, was converted to GAT and thus an EcoRV sitewas introduced into that site, to give a plasmid named pchCKB1 (FIG.45).

Then, the EcoRV site of pchCKB1 was converted to a HindIII cleavage sitein the following manner.

Thus, 2 μg of the plasmid pchCKB1 was added to 10 μl of 100 mMTris-hydrochloride buffer (pH 7.5) containing 6 mM magnesium chlorideand 100 mM sodium chloride, 10 units of EcoRI was further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wassubjected to phenol-chloroform extraction and then to ethanolprecipitation, the precipitate was dissolved in a total of 40 μl of DNApolymerase I buffer, 5 units of Escherichia coli-derived polymerase IKlenow fragment was added, and the reaction was carried out at 37° C.for 30 minutes for rendering the 5' protruding ends formed upon EcoRIdigestion blunt-ended. The reaction mixture was subjected tophenol-chloroform extraction and then ethanol precipitation, theprecipitate was dissolved, together with 0.1 μg of a HindIII linker(Takara Shuzo), in 20 μl of T4 ligase buffer; 350 units of T4 ligase wasadded to the solution, and ligation was effected at 4° C. for 24 hours.The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli HB101 and the plasmid pchCKC1 shown in FIG. 46 wasobtained.

5. Construction of vectors for chimeric human antibody H chainexpression

(1) Construction of a vector to be used in constructing chimeric humanantibody H chain expression vectors (vector for chimeric human antibodyH chain expression) The plasmid pIg1SE1d4 obtained in Paragraph 2 (11)(2 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 6 mM magnesium chloride and 100 mM sodium chloride, 10 unitseach of EcoRV and ApaI were further added, and digestion was effected at37° C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 9.2 kb)resulting from cleavage of pIg1SE1d4 with EcoRV and ApaI was recovered.

Then, 2 μg of pPCVHhCGI1 obtained in Paragraph 4 (2) was added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mM magnesiumchloride, 10 units of ApaI and 10 units of SmaI were further added, anddigestion was conducted at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of a DNAfragment (about 1 kb) resulting from cleavage of pPCVHhCGI1 with ApaIand SmaI and containing the human immunoglobulin H chain constant regiongene was recovered.

Then, 0.1 μg of the EcoRV-ApaI fragment (about 9.2 kb) of pIg1SE1d4 asobtained above and 0.1 μg of the ApaI-SmaI fragment (about 1 kb) ofpPCVHhCG1 as obtained above were dissolved in a total of 20 μl of T4ligase buffer; 350 units of T4 DNA ligase was added to the solution, andligation was conducted at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe vector pCHiIgHB2 for chimeric human antibody H chain expression asshown in FIG. 47 was obtained.

(2) Construction of a vector to be used in constructing chimeric humanantibody L chain expression vectors (vector for chimeric human antibodyL chain expression)

The plasmid pIg1SE1d4 obtained in Paragraph 2 (11) (2 μg) was added to30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 6 mMmagnesium chloride and 100 mM sodium chloride, 10 units of EcoRV and 10units of HindIII were further added, and digestion was effected at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 1.5 μg of a DNA fragment (about 9.2 kb)resulting from cleavage of pIg1SE1d4 with EcoRV and HindIII wasrecovered.

Then, 2 μg of pckCKC1 obtained in Paragraph 4 (3) was added to 30 μl of10 mM Tris-hydrochloride (pH 7.5) containing 6 mM magnesium chloride and100 mM sodium chloride, 10 units of EcoRV and 10 units of HindIII werefurther added, and digestion was carried out at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.2 μg of a DNA fragment (about 0.6 kb) resulting from cleavage ofpPCVLhCK1 with EcoRV and HindIII and containing the human immunoglobulinL chain constant region gene was recovered.

Then, 0.1 μg of the EcoRV-HindIII fragment (about 9.2 kb) of pIg1SE1d4as obtained above and 0.1 μg of the EcoRV-HindIII fragment (about 0.6kb) of pchCKC1 as obtained above were dissolved in a total of 20 μl ofT4 ligase buffer, 350 units of T4 DNA ligase was added to the solution,and ligation was carried out at 4° C. for 24 hours. The thus-obtainedrecombinant plasmid DNA was used to transform Escherichia coli HB101 andthe vector pChiIgLA1 for chimeric human antibody L chain expression asshown in FIG. 48 was obtained.

REFERENCE EXAMPLE 2

Construction of a chimeric human antibody H chain expression vector,pChi641HA1

1. Isolation of mRNA from mouse anti-GD₃ monoclonal antibodyKM-641-producing hybridoma cells

Using mRNA extraction kit Fast Track (product number K1593-02)manufactured by Invitrogen, 34 μg of mRNA was isolated from 1×10⁸ mouseanti-GD₃ monoclonal antibody KM-641-producing hybridoma cells obtainableas in Reference Example 1.

2. Construction of a KM-641 H chain cDNA library and a KM-641 L chaincDNA library

Using 3 μg of the mRNA obtained in Paragraph 1 and using cDNA synthesiskit ZAP-cDNA Synthesis Kit (product number sc200400) manufactured byStratagene, cDNA having an EcoRI adapter at the 5' terminus and cDNAhaving an XhoI adapter at the 3' terminus were synthesized. About 6 μgof each cDNA was dissolved in 10 μl of sterilized water and fractionatedby agarose gel electrophoresis. In this way, about 0.1 μg of a cDNAfragment having a size of about 1.8 kb and corresponding to the H chainand a cDNA fragment having a size of about 1.0 kb and corresponding tothe L chain were recovered. Then, 0.1 μg of the cDNA fragment of about1.8 kb in size, 0.1 μg of the cDNA fragment of about 1.0 kb in size and1 μg of Uni-ZAP XR (Stratagene; derived from the Lambda ZAPII vector bycleavage with EcoRI and XhoI, followed by treatment with calf intestinealkaline phosphatase), to be used as the vector, were dissolved in T4ligase buffer; 175 units of T4 DNA ligase was added, and the mixture wasincubated at 12° C. for 10 hours and further at room temperature for 2hours. A 4-μl portion of the reaction mixture was packaged into thelambda phage by the conventional method Maniatis et al. (ed.): MolecularCloning, 1989, p. 2.95! using Giga Pak Gold (Stratagene), followed bytransfection of Escherichia coli PLK-F with the packaging mixture by theconventional method Maniatis et al. (ed.): Molecular Cloning, 1989, p.2.95-107!. As an H chain cDNA library and as an L chain cDNA library,about 10,000 phage clones were respectively obtained. The phages werethen fixed onto nitrocellulose filters by the conventional methodManiatis et al. (ed.): Molecular Cloning, 1989, p. 2.112!.

3. Cloning of the monoclonal antibody KM-641 H chain and L chain cDNAs

Using probes prepared by labeling a mouse Cγ1 gene (mouse immunoglobulinconstant region chromosomal gene)-containing EcoRI fragment (about 6.8kb) Roeder et al.: Proc. Natl. Acad. Sci. U.S.A., 78, 474 (1981)! and amouse Cκ gene-containing HindIII-BamHI fragment (about 3 kb) Sakano etal.: Nature, 280, 288 (1979)! with ³² p, one phage clone stronglyassociable with the former probe at 65° C. and one phage clone stronglyassociable with the latter probe at 65° C. were isolated from the Hchain cDNA library and L chain cDNA library constructed in Paragraph 2in accordance with the conventional method Maniatis et al. (ed.):Molecular Cloning, 1989, p. 2.108!. Then, by converting the phage clonesto pBluescript plasmids using cDNA synthesis kit ZAP-cDNA Synthesis Kit(product number sc200400) manufactured by Stratagene, a KM-641 H chaincDNA-containing recombinant plasmid, pKM641HA3, and a KM-641 L chaincDNA-containing recombinant plasmid, pKM641LA2, were obtained. Cleavageof pKM641HA3 and pKM641LA2 with EcoRI and XhoI revealed that a cDNAfragment of about 1.6 kb and a cDNA fragment of about 0.9 kb had beeninserted therein, respectively (FIG. 49).

4. Base sequences of the immunoglobulin variable regions in the KM-641 Hchain cDNA (pKM641HA3) and KM-641 L chain cDNA (pKM641LA2)

The base sequences of the immunoglobulin regions in pKM641HA3 andpKM641LA2 obtained in Paragraph 3 were determined by the dideoxy methodManiatis et al. (ed.): Molecular Cloning, 1989, p. 13.42! usingSequenase Version 2.0 DNA Sequencing Kit (United States BiochemicalCorporation). The results obtained are shown in SEQ ID NO:19 and SEQ IDNO:20. In pKM641LA2, a methionine codon, presumably the initiation codonATG, was found in the vicinity of the 5' terminus and the cDNA was aleader sequence-containing full-length one. In pKM641HA3, no methionineinitiation codon was found and the leader sequence was partly lacking.

5. Construction of a KM-641-derived chimeric human antibody H chainexpression vector

A chimeric human antibody H chain expression vector was constructed byjoining the H chain variable region gene obtained by cleaving theplasmid pKM641HA3 at the AluI site near the 5' terminus of the variableregion gene and at the StyI site near the 3' terminus of the variableregion gene to the vector for chimeric human antibody H chain expressionas obtained in Reference Example 1 using the synthetic DNAs defined bySEQ ID NO:21 and SEQ ID NO:22 (FIG. 50).

First, the DNA defined by SEQ ID NO:22 composed of the base sequencefrom the 3' terminus of the immunoglobulin H chain variable region inpKM641HA3 to the StyI cleavage site near said 3' terminus and the basesequence from the 5' terminus of the immunoglobulin H chain constantregion in pAGE28 to the ApaI cleavage site near said 5' terminus andhaving a StyI cleavage site and an ApaI cleavage site on the respectivetermini (cf. FIG. 50) was synthesized using a DNA synthesizer. Thissynthetic DNA was then introduced into the plasmid pKM641HA3 in thefollowing manner.

Three μg of pKM641HA3 was added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodiumchloride and 1 mM DTT, 10 units of EcoRI and 10 units of StyI werefurther added, and digestion was effected at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.3 μg of a 0.41 kb DNA fragment was recovered. Then, 3 μg ofpAGE28 Mizukami et al.: J. Biochem., 101, 1307-1310 (1987)! was added to30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mMmagnesium chloride and 6 mM 2-mercaptoethanol, 10 units of EcoRI and 10units of ApaI were further added, and digestion was carried out at 37°C. for 4 hours. The reaction mixture was fractionated by agarose gelelectrophoresis and about 2 μg of a 2.45 kb DNA fragment was recovered.Then, 0.1 μg of the EcoRI-StyI fragment (about 0.41 kb) of pKM641HA3, asobtained above, 0.1 μg of the EcoRI-ApaI fragment (about 2.45 kb) ofpAGE28, as obtained above, and 0.3 μg of the synthetic DNA, defined bySEQ ID NO:22, were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wasconducted at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpKM641HE1 shown in FIG. 51 was obtained.

Since pKM641HE1 had no leader sequence, the following measure was takento supplement the deficit using the synthetic DNA defined by SEQ IDNO:21.

pKM641HE1 (3 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 7 mM magnesium chloride and 6 mM 2-mercaptoethanol,10 units of EcoRI and 10 units of ApaI were further added, and digestionwas effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.4 μg of a DNAfragment of about 0.42 kb in size was recovered. The EcoRI-ApaI fragment(about 0.42 kb; 0.4 μg) of pKM641HE1 was added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 7 mM magnesium chloride,50 mM sodium chloride and 6 mM 2-mercaptoethanol, 10 units of AluI wasfurther added, and digestion was effected at 37° C. for 4 hours. Thereaction mixture was subjected to phenol-chloroform extraction and thento ethanol precipitation, and about 0.3 μg of a DNA fragment of about0.4 kb in size was recovered.

Then, 0.1 μg of the AluI-ApaI fragment (about 0.4 kb) of pKM641HE1 asobtained above, 0.1 μg of the EcoRI-ApaI fragment (about 2.45 kb) ofpAGE28 as obtained above and 0.3 μg of the synthetic DNA defined by SEQID NO:21 were dissolved in a total of 20 μl of T4 ligase buffer; 350units of T4 ligase was added to the solution, and ligation was carriedout at 4° C. for 24 hours. The thus-obtained recombinant plasmid DNA wasused to transform Escherichia coli HB101 and the plasmid pKM641HF1 shownin FIG. 52 was obtained.

Then, the immunoglobulin H chain variable region of pKM641HF1 wasintroduced into the vector pChiIgHB2 for chimeric human antibody H chainexpression, as follows.

pKM641HF1 (3 μg) was added to 30 μl of 10 mM Tris-hydrochloride buffer(pH 7.5) containing 7 mM magnesium chloride and 6 mM 2-mercaptoethanol,10 units of EcoRI and 10 units of ApaI were further added, and digestionwas effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.5 μg of a 0.44kb DNA fragment was recovered. Then, 3 μg of pChiIgHB2 was added to 30μl of 10 mM Tris-hydrochloride buffer (pH 7.5) containing 7 mM magnesiumchloride and 6 mM 2-mercaptoethanol, 10 units of EcoRI and 10 units ofApaI were further added, and digestion was conducted at 37° C. for 4hours. The reaction mixture was subjected to phenol-chloroformextraction and about 3 μg of DNA was recovered. Then, 0.1 μg of theEcoRI-ApaI fragment (about 0.44 kb) of pKM641HF1 as obtained above and0.1 μg of the EcoRI-ApaI fragment (about 10.1 kb) of pChiIgHB2 asobtained above were dissolved in a total of 20 μl of T4 ligase buffer;350 units of T4 ligase was added to the solution, and ligation wascarried out at 4° C. for 24 hours. The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 and the plasmidpChi641HA1 shown in FIG. 53 was obtained.

Then, the KM50-derived immunoglobulin H chain promoter and enhancerregion of pChi641HA1 was replaced with MoLTR, as follows.

pChi641HA1 (3 μg) was added to 30 μl of 50 mM Tris-hydrochloride buffer(pH 7.5) containing 10 mM magnesium chloride, 50 mM sodium chloride and1 mM DTT, 10 units of EcoRI and 10 units of XhoI were further added, anddigestion was effected at 37° C. for 4 hours. The reaction mixture wasfractionated by agarose gel electrophoresis and about 0.2 μg of a DNAfragment of about 8.8 kb in size was recovered. pPMOL3 (3 μg) obtainedin Example 1, Paragraph 2 was added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 50 mM sodiumchloride and 1 mM DTT; 10 units of EcoRI and 10 units of XhoI werefurther added, and digestion was carried out at 37° C. for 4 hours. Thereaction mixture was fractionated by agarose gel electrophoresis andabout 0.3 μg of a MoLTR-containing DNA fragment (0.63 kg) was recovered.Then, 0.1 μg of the EcoRI-XhoI fragment of pChi641HA1 and 0.1 μg of theEcoRI-XhoI fragment of pPMOL3 were dissolved in 20 μl of T4 ligasebuffer, 175 units of T4 DNA ligase was added, and the mixture wasincubated at 4° C. for 1 day. The reaction mixture was used to transformEscherichia coli MB101 and the KM-641-derived chimeric human H chainexpression vector pChi641HAM1 shown in FIG. 54 was obtained.

EXAMPLE 2 Production of human CDR-transplanted anti-GM₂ antibodies

1. Construction of DNAs each coding for human CDR-transplanted anti-GM₂antibody H chain variable region and human CDR-transplanted anti-GM₂antibody L chain variable region

(1) Construction of DNA coding for human CDR-transplanted anti-GM₂antibody H chain variable region

A DNA coding for a human CDR-tansplanted anti-GM₂ antibody H chainvariable region, hKM796H, which contains amino acid sequences of SEQ IDNO:6, SEQ ID NO:7 and SEQ ID NO:8, was constructed in the followingmanner.

NEWM BIO/TECHNOLOGY, 9, 266 (1991)! was used as human antibody H chainvariable region-encoding DNA to which each CDR was to be transplanted.DNAs set forth in SEQ ID NO:23 through NO:29 corresponding to NEWM inwhich each CDR was replaced with amino acid sequences of SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8 were synthesized using an automatic DNAsynthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.).The thus-obtained synthetic DNAs (50 picomoles each) were dissolved in20 μl of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4polynucleotide kinase was added, and 5'-phosphorylation was carried outat 37° C. for 30 minutes. Ten picomoles each of the resultingphosphorylated synthetic DNAs, which had restriction enzyme sites onboth ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:23 throuthNO:29) using a DNA ligation kit (Takara Shuzo) in accordance with themanufacturer's instruction attached to the kit to obtain a DNA, hKM796H,shown in FIG. 55. The amino acid sequence corresponding to hKM796H isshown in SEQ ID NO:36.

(2) Construction of DNA coding for human CDR-transplanted anti-GM₂antibody L chain variable region

A DNA coding for a human CDR-transplanted anti-GM₂ antibody L chainvariable region, hKM796L, which contains amino acid sequences of SEQ IDNO:9, SEQ ID NO:10 and SEQ ID NO:11, was constructed in the followingmanner.

REI BIO/TECHNOLOGY, 9, 266 (1991)! was used as human antibody L chainvariable region-encoding DNA to which each CDR was to be transplanted.DNAs set forth in SEQ ID NO:30 through NO:35 corresponding to REI inwhich each CDR was replaced with amino acid sequences of SEQ ID NO:9,SEQ ID NO:10 and SEQ ID NO:11 were synthesized using an automatic DNAsynthesizer (model 380A manufactured by Applied Biosystems Co., Ltd.).The thus-obtained synthetic DNAs (50 picomoles each) were dissolved in20 μI of 50 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mMmagnesium chloride, 5 mM DTT, 0.1 mM EDTA and 0.5 mM ATP, 5 units of T4polynucleotide kinase was added, and 5'-phosphorylation was carried outat 37° C. for 30 minutes. Ten picomoles each of the resultingphosphorylated synthetic DNAs, which had restriction enzyme sites onboth ends, were ligated in the order of SEQ ID NO. (SEQ ID NO:30 throughNO:35) using a DNA ligation kit (Takara Shuzo) in accordance with themanufacturer's instruction attached to the kit to obtain a DNA, hKM796L,shown in FIG. 56. Tho amino acid sequence corresponding to hKM796L isshown in SEQ ID NO:37.

2. Construction of human CDR-transplanted antibody H chain expressionvector and human CDR-transplanted antibody L chain expression vector

(1) Construction of human CDR-transplanted antibody H chain expressionvector

A NotI-ApaI fragment of the DNA coding for human CDR-transplantedantibody H chain variable region, obtained in Paragraph 1(1) of Example2, was ligated to the plasmid pChi796HM1, obtained in Paragraph 7(3) ofExample 1, in the following manner (FIG. 57).

Three μg of pChi796HM1, obtained in Paragraph 7(3) of Example 1, weredissolved in 30 μl of 10 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride and 1 mM DTT, 10 units of ApaI wereadded thereto and the mixture was allowed to react at 37° C. for 1 hour.The resulting mixture was subjected to ethanol precipitation and thethus-obtained precipitate was dissolved in 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Ten units of NotI were addedthereto to allow the mixture to react at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis to recover about2 μg of a DNA fragment of about 9.0 kb. Then, about 0.1 μg of thethus-obtained ApaI-NotI fragment of pChi796HM1 was ligatod to 0.5 pmolesof the NotI-ApaI fragment of the DNA coding for human CDR-transplantedantibody H chain variable region, obtained in Paragraph 1(1) of Example2, using a DNA ligation kit (Takara Shuzo). The resulting recombinantplasmid DNA was used to transform Escherichia coli HB101 and the plasmidphKM796H2l shown in FIG. 57 was obtained.

Then, a human CDR-transplanted antibody H chain expression vector wasconstructed by introducing β-globulin 3' splicing signal into theplasmid phKM796HM1 in the following manner (FIG. 58).

Three μg of phKM796HM1 were added to 30 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 1 unitof KpnI was added thereto. The mixture was allowed to react at 37° C.for 10 minutes to effect partial digestion. The resulting mixture wassubjected to ethanol precipitation and the thus-obtained precipitate wasdissolved in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)Containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mMDTT. After adding 1 unit of XhoI, the mixture was allowed to react at37° C. for 10 minutes to effect partial digestion. The reaction mixturewas fractionated by agarose gel electrophoresis to recover about 0.2 μgof a DNA fragment of about 2.1 kb. Separately, 3 μg of pAGE148, obtainedin Paragraph 7(2) of Example 1, were added to 30 μl of 10 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chlorideand 1 mM DTT. Ten units of KpnI were added thereto to allow the mixtureto react at 37° C. for 1 hour. The reaction mixture was subjected toethanol precipitation and the thus-obtained precipitate was dissolved in30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT. After adding 10units of XhoI, the mixture was allowed to react at 37° C. for 1 hour andthen fractionated by agarose gel electrophoresis to recover about 1 μgof a DNA fragment of about 8.7 kb. One tenth μg of the thus-obtainedXhoI-KpnI fragment of phKM796HM1 was ligated to 0.1 μg of the XhoI-KpnIfragment of pAGE148 using a DNA ligation kit (Takara Shuzo). Thethus-obtained recombinant plasmid DNA was used to transform Escherichiacoli HB101 to obtain the plasmid phKM796HMS1 shown in FIG. 58.

(2) Construction of human CDR-transplanted antibody L chain expressionvector

An EcoRI fragment having blunt ends of the DNA coding for humanCDR-transplanted antibody L chain variable region, obtained in Paragraph1 (2) of Example 2 was ligated to the chimeric human antibody L chainexpression vector pChiIgLA1 in the following manner (FIG. 59).

Three μg of pChiIgLA1, obtained in Reference Example 1, were added to 30μg of 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT, 10 units ofEcoRI and 10 units of EcoRV were added thereto and tho mixture wasallowed to react at 37° C. for 1 hour. The reaction mixture wasfractionated by agarose gel electrophoresis to recover about 1 μg of aDNA fragment of about 8.6 kb. Then, about 0.1 μg of the thus-obtainedEcoRI-EcoRV fragment of pChiIgLA1 Was ligated to 0.5 pmoles of the EcoRIfragment having blunt ends derived from the DNA coding for humanCDR-transplanted antibody L chain variable region, obtained in Paragraph1(2) of Example 2, using a DNA ligation kit (Takara Shuzo). Theresulting recombinant plasmid DNA was used to transform Escherichia coilHB101 and the plasmid phKM796LI1 shown in FIG. 59 was obtained.

Then, P_(MO) was introduced into the plasmid phKM796LI1 in the followingmanner (FIG. 60).

Three μg of phKM796LI1 were added to 30 μl of 50 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride, 100 mM sodiumchloride and 1 mM DTT, 10 units of EcoRI and 10 units of XhoI were addedthereto, and the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis torecover about 1 μg of a DNA fragment of about 8.2 kb. Separately, 3 μgof the chimeric human antibody H chain expression vector pChi641HAM1,obtained in Reference Example 2, were added to 30 μl of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT. Ten units of EcoRI and 10 units ofXhoI were added thereto to allow the mixture to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose galelectrophoresis to recover about 0.3 μg of a DNA fragment of about 0.6kb. One tenth μg of the thus-obtained EcoRI-XhoI fragment of pChi641HAM1was ligated to 0.1 μg of the EcoRI-XhoI fragment of phKM796LI1 using aDNA ligation kit (Takara Shuzo). The thus-obtained recombinant plasmidDNA was used to transform Escherichia coli HB101 to obtain the plasmidphKM796LM1 shown in FIG. 60.

Then, a human CDR-transplanted antibody L chain expression vector wasconstructed by introducing β-globulin 3' splicing signal into theplasmid phKM796LM1 in the following manner (FIG. 61).

Three μg of phKM796LM1 were added to 30 μl of 10 mM Tris-hydrochloridebuffer (pH 7.5) containing 10 mM magnesium chloride and 1 mM DTT, 10units of KpnI were added thereto, and the mixture was allowed to reactat 37° C. for 1 hour, The resulting mixture was subjected to ethanolprecipitation and the thus-obtained precipitate wag dissolved in 30 μlof 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT. After adding 10 units ofXhoI, the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis torecover about 0.3 μg of a DNA fragment of about 1.6 kb. Separately, 3 μgof pAGE148, obtained in Paragraph 7(2) of Example 1, were added to 30 μlof 10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride and 1 mM DTT. Ten units of KpnI were added thereto to allow themixture to react at 37° C. for 1 hour. The reaction mixture wassubjected to ethanol precipitation and the thus-obtained precipitate wasdissolved in 30 μl of 50 mM Tris-hydrochloride buffer (pH 7.5)containing 10 mM magnesium chloride, 100 mM sodium chloride and 1 mMDTT. After adding 10 units of XhoI, the mixture was allowed to react at37° C. for 1 hour and then fractionated by agarose gel electrophoresisto recover about 1 μg of a DNA fragment of about 8.7 kb. One tenth μg ofthe thus-obtained XhoI-KpnI fragment of phKM796LM1 was ligated to 0.1 μgof the XhoI-KpnI fragment of pAGE148 using a DNA ligation kit (TakaraShuzo). The thus-obtained recombinant plasmid DNA was used to transformEscherichia coli HB101 to obtain the plasmid phKM796LMS1 shown in FIG.61.

3. Construction of human CDR-transplanted antibody H chain and L chaintandem expression vector

A tandem expression vector containing both of cDNA coding for humanCDR-transplanted antibody H chain and cDNA coding for humanCDR-transplanted antibody L chain was constructed in the followingmanner (FIG. 62 and FIG. 63). Three μg of phKM796HMS1, obtained inParagraph 2(1) of Example 2, were dissolved in 30 μg of 50 mMTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 1 unit of SalI was added theretoand the mixture was allowed to react at 37° C. for 10 minutes to effectpartial digestion. The resulting mixture was subjected to ethanolprecipitation and the thus-obtained precipitate was dissolved in 30 μlof 50 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT. Ten units of MluI wasadded thereto to allow the mixture to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis torecover about 0.2 μg of a DNA fragment of about 5.9 kb. Then, about 2 μqof pAGE107 as described in EP-A-0 405 285 was added to 30 μl of 50 mMTrig-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of MluI and 10 units ofSalI were added thereto, and the mixture was allowed to react at 37° C.for 1 hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.2 μg of a DNA fragment of about 3.35kb. Then, 0.1 μg of the thus-obtained MluI-SalI fragment of phKM796HMS1was ligated to 0.1 μg of the MluI-SalI fragment of pAGE107 using a DNAligation kit (Takara Shuzo). The thus-obtained recombinant plasmid DNAwas used to transform Escherichia coli HB101to obtain the plasmidphKM796H107 shown in FIG. 62.

Then, 3 μg of phKM796H107 were added to 30 μl of 10 mMTris-hydrochloride (pH 7.5) containing 10 mM magnesium chloride, 100 mMsodium chloride and 1 mM DTT, 10 units of ClaI was added thereto and themixture was allowed to react at 37° C. for 1 hour. The reaction mixturewas subjected to phenol-chloroform extraction and ethanol precipitation.The resulting precipitate was dissolved in 20 μg of DNA polymerase Ibuffer, 5 units of Escherichia coli-derived DNA polymerase I Klenowfragment were added, and the 5' cohesive ends produced by ClaI digestionwere rendered blunt by incubation at 22° C. for 30 minutes. The reactionmixture was fractionated by agarose gel electrophoresis to recover about0.2 μg of a DNA fragment of about 3.35 kb. The reaction mixture was alsosubjected to phenol-chloroform extraction and then to ethanolprecipitation. The resulting precipitate was dissolved in 30 μl of 10 inTris-hydrochloride buffer (pH 7.5) containing 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, 10 units of MluI were added theretoand the mixture was allowed to react at 37° C. for 1 hour. The reactionmixture was fractionated by agarose gel electrophoresis to recover about0.3 μg of a DNA fragment of about 7.5 kb. Separately, 3 μg ofphKM796LMS1 were added to 30 μl of 50 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM magnesium chloride, 100 mM sodium chloride and 1mM DTT, 10 units of XhoI were added and the mixture was allowed to reactat 37° C. for 1 hour. The reaction mixture was subjected tophenol-chloroform extraction and then to ethanol precipitation. Theresulting precipitate was dissolved in 20 μl of DNA polymerase I buffer,5 units of Escherichia coli-derived DNA polymerase I Klenow fragmentwere added, and the 5' cohesive ends produced by XhoI digestion weretendered blunt by incubation at 22° C. for 30 minutes. The reactionmixture was subjected to phenol-chloroform extraction followed byethanol precipitation. The resulting precipitate was added to 30 μl of10 mM Tris-hydrochloride buffer (pH 7.5) containing 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT, 10 units of MluI wereadded thereto, and the mixture was allowed to react At 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis to recover about 0.3 μg of a DNA fragment of about 9.3kb. Then, 0.1 μg of the thus-obtained MluI-ClaI fragment of phKM796H107was ligated to 0.1 μg of the MluI-XhoI fragment of phKM796LMS1 using aDNA ligation kit (Takara Shuzo). The thus-obtained recombinant plasmidDNA was used to transform Escherichia coil HB101 to obtain the plasmidphKM796HL1 shown in FIG. 63.

4. Expression of human CDR-transplanted anti-GM₂ antibody in YB2/0 cells

The plasmids were introduced into YB2/0 cells by the electroporationmethod of Miyaji et al. Cytotechnology, 3, 133 (1990)!.

After introduction of 4 μg of phKM796HL1 obtained in Paragraph 3 ofExample 2 into 4×10⁶ YB2/0 (ATCC CRL1581) cells, the cells weresuspended in 40 ml of RPMI1640-PCS(10) RPMI1640 medium (NissuiPharmaceutical) containing 10% of FCS, 1/4 volume of 7.5% NaHCO ₃, 3% of200 mM L-glutamine solution (Gibco) and 0.5% of penicillin-streptomycinsolution (Gibco; containing 5, 000 units/ml penicillin and 5,000 μg/mlstreptomycin)!, and the suspension was distributed in 200-μl portionsinto wells of 96-well microtiter plates. After 24 hours of incubation at37° C. in a CO₂ incubator, G418 (Gibco) was added to a concentration of0.5 mg/ml and then incubation was continued for 1 to 2 weeks.Transformant colonies appeared, the culture fluid was recovered fromeach well in which the cells had grown to confluence and anenzyme-linked immunosorbent assay (ELISA) described in Paragraph 11 ofExample 1 was conducted for anti-GM₂ human CDR-transplanted antibodyactivity measurement.

The clone showing the highest activity in ELISA among the clonesobtained gave a human CDR-transplanted anti-GM₂ antibody content ofabout 0.1 μg/ml of culture fluid.

Cells of the clone showing the above-mentioned human CDR-transplantedanti-GM₂ antibody activity were suspended in RPMI1640-FCS(10) mediumcontaining 0.5 mg/ml G418 and 50 nM MTX to a concentration of 1 to 2×10⁵cells/ml, and the suspension was distributed in 2-ml portions into wellsof 24-well plates. Incubation was performed at 37° C. in a CO₂ incubatorfor 1 to 2 weeks to induce 50 nM MTX-resistant clones. At the time ofconfluence the human CDR-transplanted anti-GM₂ antibody activity in eachculture fluid was determined by ELISA. The 50 nM MTX-resistant cloneshowing the highest activity among the clones obtained showed a humanCDR-transplanted anti-GM₂ antibody content of about 1.0 μg/ml.

Cells of the above 50 nm MTX-resistant clone were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX to aconcentration of 1 to 2×10⁵ cells/ml, and the suspension was distributedin 2-ml portions into wells of 24-well plates. Incubation was carriedout at 37° C. in a CO₂ incubator for 1 to 2 weeks to induce 200 nMMTX-resistant clones. At the time of confluence, each culture fluid wasassayed for human CDR-transplanted anti-GM₂ antibody activity by ELISA.The 200 nM MTX-resistant clone showing the highest activity among theclones obtained had a human CDR-transplanted anti-GM₂ antibody contentof about 5.0 μg/ml.

As described in detail hereinabove, the present invention provideshumanized antibodies reacting with the ganglioside GM₂.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 49                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: -19..-1                                                         (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN                                                  ESTABLISHED CONSENSUS                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 1"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 50..66                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 2"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 99..109                                                         (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 3"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGlyTrpSerTrpIlePheLeuPheLeuLeuSerGlyThrAlaGly                              151015                                                                        ValLeuSerGluValGlnLeuGlnGlnSerGlyProGluLeuValLys                              202530                                                                        ProGlyAlaSerValLysIleSerCysLysAlaSerGlyTyrThrPhe                              354045                                                                        ThrAspTyrAsnMetAspTrpValLysGlnSerHisGlyLysSerLeu                              505560                                                                        GluTrpIleGlyTyrIleTyrProAsnAsnGlyGlyThrGlyTyrAsn                              65707580                                                                      GlnLysPheLysSerLysAlaThrLeuThrValAspLysSerSerSer                              859095                                                                        ThrAlaTyrMetGluLeuHisSerLeuThrSerGluAspSerAlaVal                              100105110                                                                     TyrTyrCysAlaThrTyrGlyHisTyrTyrGlyTyrMetPheAlaTyr                              115120125                                                                     TrpGlyGlnGlyThrLeuValThrValSerAla                                             130135                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: -22..-1                                                         (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE TO TO AN ESTABLISHED                                      CONSENSUS                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 24..33                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 1"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 49..55                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 2"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 88..96                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 3"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetHisPheGlnValGlnIlePheSerPheLeuLeuIleSerAlaSer                              151015                                                                        ValIleMetSerArgGlyGlnIleValLeuThrGlnSerProAlaIle                              202530                                                                        MetSerAlaSerProGlyGluLysValThrIleThrCysSerAlaSer                              354045                                                                        SerSerValSerTyrMetHisTrpPheGlnGlnLysProGlyThrSer                              505560                                                                        ProLysLeuTrpIleTyrSerThrSerAsnLeuAlaSerGlyValPro                              65707580                                                                      AlaArgPheSerGlySerGlySerGlyThrSerTyrSerLeuThrIle                              859095                                                                        SerArgMetGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnArg                              100105110                                                                     SerSerTyrProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys                              115120125                                                                     Arg                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 137 amino acids                                                   (B) TYPE: amino acids                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: -19..-1                                                         (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 31..35                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 1"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 55..66                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 2"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 99..107                                                         (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISEHD                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 3"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetGlyTrpSerTrpIlePheLeuPheLeuLeuSerGlyThrAlaGly                              151015                                                                        ValLeuSerGluValGlnLeuGlnGlnSerGlyProGluLeuValLys                              202530                                                                        ProGlyAlaSerValLysIleSerCysLysAlaSerGlyTyrThrPhe                              354045                                                                        ThrAspTyrAsnMetAspTrpValLysGlnSerHisGlyLysSerLeu                              505560                                                                        GluTrpIleGlyTyrIleTyrProAsnAsnGlyGlyThrGlyTyrAsn                              65707580                                                                      GlnLysPheLysSerLysAlaThrLeuThrValAspLysSerSerSer                              859095                                                                        ThrAlaTyrMetGluLeuHisSerLeuThrSerGluAspSerAlaVal                              100105110                                                                     TyrTyrCysAlaArgAlaGlyArgTyrTyrTyrAlaTrpAspTrpGly                              115120125                                                                     GlnGlyThrLeuValThrValSerAla                                                   130135                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 405 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 10..66                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 157..171                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 1"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 214..261                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 2"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 358..369                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 3"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CATCACAGCATGGCTGTCCTGGTGCTGTTGCTCTGCCTGGTGACATTT48                            MetAlaValLeuValLeuLeuLeuCysLeuValThrPhe                                       15-10                                                                         CCAAGCTGTGTCCTGTCCCAAGTGCAGCTGAAGGAGTCAGGACCTGGT96                            ProSerCysValLeuSerGlnValGlnLeuLysGluSerGlyProGly                              51510                                                                         CTGGTGCAGCCCTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGG144                           LeuValGlnProSerGlnThrLeuSerLeuThrCysThrValSerGly                              152025                                                                        TTCTCATTAACCAGCTATACTGTAAGCTGGGTTCGCCAGCCTCCAGGA192                           PheSerLeuThrSerTyrThrValSerTrpValArgGlnProProGly                              303540                                                                        AAGGGTCTGGAGTGGATTGCAGCAATATCAAGTGGTGGAAGCACATAT240                           LysGlyLeuGluTrpIleAlaAlaIleSerSerGlyGlySerThrTyr                              455055                                                                        TATAATTCAGCTCTCAAATCACGACTGAGCATCAGCAGGGACACCTCC288                           TyrAsnSerAlaLeuLysSerArgLeuSerIleSerArgAspThrSer                              608570                                                                        AAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAAGACACA336                           LysSerGlnValPheLeuLysMetAsnSerLeuGlnThrGluAspThr                              75808590                                                                      GCCATGTACTTCTGTGCCCCTTCTGAGGGGGCCTGGGGCCAAGGAGTC384                           AlaMetTyrPheCysAlaProSerGluGlyAlaTrpGlyGlnGlyVal                              95100105                                                                      ATGGTCACAGTCTCCTCAGAG405                                                      MetValThrValSerSerGlu                                                         110                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 19..78                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 148..180                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABILSHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 1"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 226..246                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 2"                      (ix) FEATURE:                                                                 (A) NAME/KEY: domain                                                          (B) LOCATION: 343..369                                                        (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (D) OTHER INFORMATION: /product="HYPERVARIABLE REGION 3"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACAGGACACAGGTCAGTCATGATGGCTCCAGTCCAGCTCTTAGGGCTGCTG51                         MetMetAlaProValGlnLeuLeuGlyLeuLeu                                             20-15-10                                                                      CTGATTTGGCTCCCAGCCATGAGATGTGACATCCAGATGACCCAGTCT99                            LeuIleTrpLeuProAlaMetArgCysAspIleGlnMetThrGlnSer                              515                                                                           CCTTCATTCCTGTCTGCATCTGTGGGAGACAGAGTCACTATCAACTGC147                           ProSerPheLeuSerAlaSerValGlyAspArgValThrIleAsnCys                              101520                                                                        AAAGCAAGTCAGAATATTAACAAGTACTTAAACTGGTATCAGCAAAAG195                           LysAlaSerGlnAsnIleAsnLysTyrLeuAsnTrpTyrGlnGlnLys                              253035                                                                        CTTGGAGAAGCTCCCAAACGCCTGATATATAATACAAACAATTTGCAA243                           LeuGlyGluAlaProLysArgLeuIleTyrAsnThrAsnAsnLeuGln                              40455055                                                                      ACGGGCATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTAC291                           ThrGlyIleProSerArgPheSerGlySerGlySerGlyThrAspTyr                              606570                                                                        ACACTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACATATTTC339                           ThrLeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrPhe                              758085                                                                        TGCTTGCAGCATAATAGTTTTCCGAACACGTTTGGAGCTGGGACCAAG387                           CysLeuGlnHisAsnSerPheProAsnThrPheGlyAlaGlyThrLys                              9095100                                                                       CTGGAGCTGAAACGG402                                                            LeuGluLeuLysArg                                                               105                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspTyrAsnMetAsp                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TyrIleTyrProAsnAsnGlyGlyThrGlyTyrAsnGlnLysPheLys                              151015                                                                        Ser                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TyrGlyHisTyrTyrGlyTyrMetPheAlaTyr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerAlaSerSerSerValSerTyrMetHis                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      SerThrSerAsnLeuAlaSer                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlnGlnArgSerSerTyrProTyrThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ValThrValSerAlaAlaSerThrLysGly                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGAACTGTGGCT46                              ThrPheGlyGlyGlyThrLysLeuGluIleLysArgThrValAla                                 151015                                                                        GCACC51                                                                       Ala                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CAAGGAGTCATGGTCACAGTCTCGAGCGCCTCCACCAAGGGC42                                  GlnGlyValMetValThrValSerSerAlaSerThrLysGly                                    1510                                                                          C43                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CACGTTTGGAGCTGGTACCAAGCTTGAGCTCAAACGAACTGTGGCT46                              ThrPheGlyAlaGlyThrLysLeuGluLeuLysArgThrValAla                                 151015                                                                        GCACC51                                                                       Ala                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 812 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: HYBRIDOMA KM50                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: TATA.sub.-- signal                                              (B) LOCATION: 261..267                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AAAGTCAGACAACTTTGTAGAGTAGGTTCTATCAATCCTACTGCAATCCAACATCACTGA60                GGACAAATGTTTATACTGAGGAACCTGGTCTTGTGTGATACGTACTTTCTGTGGGAAGCA120               GATACGCACTCTCATGTGGCTCCTGAATTTCCCATCACAGAATGATACATCTTGAGTCCT180               AAAATTTAAGTACACCATCAGTGTCAGCACCTGGTGAGGAAATGCAAATCTCTCCTGGAT240               CCACCCAACCTTGGGTTGAAAAGCCAAAGCTGGGCCTGGGTACTCACTGGTGTGCAGCC299                ATGGACAGGCTTACTTCCTCATTCCTACTGCTGATGGTCCCTGCA344                              MetAspArgLeuThrSerSerPheLeuLeuLeuMetValProAla                                 15-10-5                                                                       TGTGAGTACCAAAGCTTCCTAAGTGATGAACTGTTCTATCCTCACCTGTTCAAACCTGAC404               CTCCTCCCCTTTGATTTCTCCACAGATGTCCTGTCTCAGGTTACTCTGAAA455                        TyrValLeuSerGlnValThrLeuLys                                                   15                                                                            GAATCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACT503                           GluSerGlyProGlyIleLeuGlnProSerGlnThrLeuSerLeuThr                              101520                                                                        TGCTCTTTCTCTGGGTTTTCACTGAGCACTTATGGTATGTGTGTGGGC551                           CysSerPheSerGlyPheSerLeuSerThrTyrGlyMetCysValGly                              253035                                                                        TGGATTCGTCAGTCTTCAGGGAAGGGTCTGGAGTGGCTGGCAAACGTT599                           TrpIleArgGlnSerSerGlyLysGlyLeuGluTrpLeuAlaAsnVal                              404550                                                                        TGGTGGAGTGATGCTAAGTACTACAATCCATCTCTGAAAAACCGGCTC647                           TrpTrpSerAspAlaLysTyrTyrAsnProSerLeuLysAsnArgLeu                              556065                                                                        ACAATCTCCAAGGACACCTCCAACAACCAAGCATTCCTCAAGATCACC695                           ThrIleSerLysAspThrSerAsnAsnGlnAlaPheLeuLysIleThr                              70758085                                                                      AATATGGACACTGCAGATACTGCCATATACTACTGTGCTGGGAGAGGG743                           AsnMetAspThrAlaAspThrAlaIleTyrTyrCysAlaGlyArgGly                              9095100                                                                       GCTACGGAGGGTATAGTGAGCTTTGATTACTGGGGCCACGGAGTCATG791                           AlaThrGluGlyIleValSerPheAspTyrTrpGlyHisGlyValMet                              105110115                                                                     GTCACAGTCTCCTCAGGTAAG812                                                      ValThrValSerSer                                                               120                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AGCTGAATTCGGGCCCGATATCAAGCTTGTCGACTCTAGAGGTACC46                              (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GATGAAGACAGATATCGCAGCCACAGTTC29                                               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 408 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: HYBRIDOMA KM-641                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 25..84                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AATTCGGCACGAGTCAGCCTGGACATGATGTCCTCTGCTCAGTTCCTTGGT51                         MetMetSerSerAlaGlnPheLeuGly                                                   20-15                                                                         CTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACA99                            LeuLeuLeuLeuCysPheGlnGlyThrArgCysAspIleGlnMetThr                              10- 515                                                                       CAGACTGCATCCTCCCTGCCTGCCTCTCTGGGAGACAGAGTCACCATC147                           GlnThrAlaSerSerLeuProAlaSerLeuGlyAspArgValThrIle                              101520                                                                        AGTTGCAGTGCAAGTCAGGACATTAGTAATTATTTAAACTGGTATCAA195                           SerCysSerAlaSerGlnAspIleSerAsnTyrLeuAsnTrpTyrGln                              253035                                                                        CAGAAACCAGATGGAACTGTTAAACTCCTGATCTTTTACTCATCAAAT243                           GlnLysProAspGlyThrValLysLeuLeuIlePheTyrSerSerAsn                              404550                                                                        TTACACTCGGGAGTCCCATCAAGGTTCAGTGGCGGTGGGTCCGGGACA291                           LeuHisSerGlyValProSerArgPheSerGlyGlyGlySerGlyThr                              556065                                                                        GATTATTCTCTCACCATCAGCAACCTGGAGCCTGAAGATATTGCCACT339                           AspTyrSerLeuThrIleSerAsnLeuGluProGluAspIleAlaThr                              70758085                                                                      TACTTTTGTCATCAGTATAGTAAGCTTCCGTGGACGTCCGGTGGAGGC387                           TyrPheCysHisGlnTyrSerLysLeuProTrpThrSerGlyGlyGly                              9095100                                                                       ACCAAGCTGGAAATCAAACGG408                                                      ThrLysLeuGluIleLysArg                                                         105                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 403 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: HYBRIDOMA KM-641                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 14..43                                                          (C) IDENTIFICATION METHOD: BY SIMILARITY                                      WITH KNOWN SEQUENCE OR TO AN ESTABLISHED                                      CONSENSUS                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AATTCGGCACGAGCTTGTCCTTGTTTTCAAAGGTGTTCAGTGTGAAGTG49                           LeuValLeuValPheLysGlyValGlnCysGluVal                                          10-51                                                                         ACGCTGGTGGAGTCTGGGGGAGACTTTGTGAAACCTGGAGGGTCCCTG97                            ThrLeuValGluSerGlyGlyAspPheValLysProGlyGlySerLeu                              51015                                                                         AAAGTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTCATTATGCCATG145                           LysValSerCysAlaAlaSerGlyPheAlaPheSerHisTyrAlaMet                              202530                                                                        TCTTGGGTTCGCCAGACTCCGGCGAAGAGGCTGGAATGGGTCGCATAT193                           SerTrpValArgGlnThrProAlaLysArgLeuGluTrpValAlaTyr                              35404550                                                                      ATTAGTAGTGGTGGTAGTGGCACCTACTATTCAGACAGTGTAAAGGGC241                           IleSerSerGlyGlySerGlyThrTyrTyrSerAspSerValLysGly                              556065                                                                        CGATTCACCATTTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAA289                           ArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyrLeuGln                              707580                                                                        ATGCGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTTCTGTACAAGA337                           MetArgSerLeuArgSerGluAspSerAlaMetTyrPheCysThrArg                              859095                                                                        GTTAAACTGGGAACCTACTACTTTGACTCCTGGGGCCAAGGCACCACT385                           ValLysLeuGlyThrTyrTyrPheAspSerTrpGlyGlnGlyThrThr                              100105110                                                                     CTCACTGTCTCCTCAGCT403                                                         LeuThrValSerSerAla                                                            115120                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AATTCACCATGGAGTTTGGGCTCAGCTGGCTTTTT35                                         MetGluPheGlyLeuSerTrpLeuPhe                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CAAGGTACCACGTTAACTGTCTCCTCAGCCTCCACCAAGGGC42                                  GlnGlyThrThrLeuThrValSerSerAlaSerThrLysGly                                    1510                                                                          C43                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGCCGCACCATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCTGGTGTC60                CTCTCTCAGGTCCAACTGCA80                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GGAGAGCGGTCCAGGTCTTGTGAGGCCTAGCCAGACCCTGAGCCTGACCTGCACCGTGT59                 (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CCGGATTCACCTTCAGCGACTACAACATGGACTGGGTGAGACAGCCACCTGGACGAGGTC60                (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCGAGTGGATTGGATATATTTATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCA60                AGAGCAGAGTGACAATGCTGG81                                                       (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TCGACACCAGCAAGAACCAGTTCAGCCTGAGACTCAGCAGCGTGACAGCCGCCGACACCG60                C61                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GGTCTATTATTGTGCGCGCTACGGTCATTACTACGGCTACATGTTTGCTTACTGGGGTCA60                AGGTAC66                                                                      (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CACCGTCACAGTCTCCTCAGCCTCCACCAAGGGCC35                                         (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AATTCACCATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCA60                TAATGTCCAGAGGAGAT77                                                           (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      ATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCTAGCGTGGGTGACAGAGTGACCATG60                AC62                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTATCAGCAGAAGCCAGGTAAGGC60                TCCAA65                                                                       (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AGCTTCTGATCTACAGCACATCCAACCTGGCTTCTGGTGTGCCAT45                               (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CTAGATTCAGCGGTAGCGGTAGCGGTACAGACTTCACCTTCACCATCAGCAGCCTCCAGC60                CAGAGGACATCGCTAC76                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid, synthetic DNA                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GTACTACTGCCAGCAAAGGAGTAGTTACCCGTACACGTTCGGCGGGGGGACCAAGGTGGA60                AATCAAACGTACGGTGGCTGCACC84                                                    (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 144 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      MetGlyTrpSerTrpIlePheLeuPheLeuLeuSerGlyThrAlaGly                              151015                                                                        ValLeuSerGlnValGlnLeuGlnGluSerGlyProGlyLeuValArg                              202530                                                                        ProSerGlnThrLeuSerLeuThrCysThrValSerGlyPheThrPhe                              354045                                                                        SerAspTyrAsnMetAspTrpValArgGlnProProGlyArgGlyLeu                              505560                                                                        GluTrpIleGlyTyrIleTyrProAsnAsnGlyGlyThrGlyTyrAsn                              65707580                                                                      GlnLysPheLysSerArgValThrMetLeuValAspThrSerLysAsn                              859095                                                                        GlnPheSerLeuArgLeuSerSerValThrAlaAlaAspThrAlaVal                              100105110                                                                     TyrTyrCysAlaArgTyrGlyHisTyrTyrGlyTyrMetPheAlaTyr                              115120125                                                                     TrpGlyGlnGlyThrThrValThrValSerSerAlaSerThrLysGly                              130135140                                                                     (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 133 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      MetHisPheGlnValGlnIlePheSerPheLeuLeuIleSerAlaSer                              151015                                                                        ValIleMetSerArgGlyAspIleGlnLeuThrGlnSerProSerSer                              202530                                                                        LeuSerAlaSerValGlyAspArgValThrIleThrCysSerAlaSer                              354045                                                                        SerSerValSerTyrMetHisTrpTyrGlnGlnLysProGlyLysAla                              505560                                                                        ProLysLeuLeuIleTyrSerThrSerAsnLeuAlaSerGlyValPro                              65707580                                                                      SerArgPheSerGlySerGlySerGlyThrAspPheThrPheThrIle                              859095                                                                        SerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGlnArg                              100105110                                                                     SerSerTyrProTyrThrPheGlyGlyGlyThrLysValGluIleLys                              115120125                                                                     ArgThrValAlaAla                                                               130                                                                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 449 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CTCCACAGTCCCTGAAGACACTGACTCTAACCATGGGATGGAGCTGGATCTTTCTCTTCC60                TCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGC120               TGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTG180               ACTACAACATGGACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATA240               TTTATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGAGCAAGGCCACATTGA300               CTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTGAGGACT360               CTGCAGTCTATTACTGTGCAACCTACGGTCATTACTACGGCTACATGTTTGCTTACTGGG420               GCCAAGGGACTCTGGTCACTGTCTCTGCA449                                              (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 393 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GACAAAATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATA60                ATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGG120               GAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAG180               CAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGA240               GTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGA300               ATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCGTACACG360               TTCGGAGGGGGGACCAAGCTGGAAATAAAACGG393                                          (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 443 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CTCCACAGTCCCTGAAGACACTGACTCTAACCATGGGATGGAGCTGGATCTTTCTCTTCC60                TCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGC120               TGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTG180               ACTACAACATGGACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATA240               TTTATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGAGCAAGGCCACATTGA300               CTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTGAGGACT360               CTGCAGTCTATTACTGTGCAAGAGCGGGGAGGTATTACTACGCCTGGGACTGGGGCCAAG420               GGACTCTGGTCACTGTCTCTGCA443                                                    (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GACTACAACATGGAC15                                                             (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      TATATTTATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGAGC51                         (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      TACGGTCATTACTACGGCTACATGTTTGCTTAC33                                           (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AGTGCCAGCTCAAGTGTAAGTTACATGCAC30                                              (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AGCACATCCAACCTGGCTTCT21                                                       (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      CAGCAAAGGAGTAGTTACCCGTACACG27                                                 (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 442 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GGCCGCACCATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCTGGTGTC60                CTCTCTCAGGTCCAACTGCAGGAGAGCGGTCCAGGTCTTGTGAGGCCTAGCCAGACCCTG120               AGCCTGACCTGCACCGTGTCCGGATTCACCTTCAGCGACTACAACATGGACTGGGTGAGA180               CAGCCACCTGGACGAGGTCTCGAGTGGATTGGATATATTTATCCTAACAATGGTGGTACT240               GGCTACAACCAGAAGTTCAAGAGCAGAGTGACAATGCTGGTCGACACCAGCAAGAACCAG300               TTCAGCCTGAGACTCAGCAGCGTGACAGCCGCCGACACCGCGGTCTATTATTGTGCGCGC360               TACGGTCATTACTACGGCTACATGTTTGCTTACTGGGGTCAAGGTACCACCGTCACAGTC420               TCCTCAGCCTCCACCAAGGGCC442                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 409 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AATTCACCATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCA60                TAATGTCCAGAGGAGATATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCTAGCGTGG120               GTGACAGAGTGACCATCACGTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTATC180               AGCAGAAGCCAGGTAAGGCTCCAAAGCTTCTGATCTACAGCACATCCAACCTGGCTTCTG240               GTGTGCCATCTAGATTCAGCGGTAGCGGTAGCGGTACAGACTTCACCTTCACCATCAGCA300               GCCTCCAGCCAGAGGACATCGCTACGTACTACTGCCAGCAAAGGAGTAGTTACCCGTACA360               CGTTCGGCGGGGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACC409                          (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      AAACGAACTGTGGCTGCACCATCTGTC27                                                 __________________________________________________________________________

What is claimed is:
 1. A chimeric antibody comprising a heavy chainvariable region and a light chain variable region of a non humanantibody and a heavy chain constant region and a light chain constantregion of a human antibody wherein said chimeric human antibody isspecific for the ganglioside GM₂ and wherein said heavy chain variableregion has an amino acid sequence having amino acids 1-120 of SEQ ID NO.1 and said light chain variable region has an amino acid sequence havingamino acids 1-107 of SEQ ID NO.
 2. 2. The chimeric antibody as claimedin claim 1, wherein said chimeric antibody is KM966 secreted bytransformant FERM BP-3931.
 3. A composition comprising the chimericantibody of claim 1 and a pharmaceutically acceptable carrier.